Metabolically engineered microbial cell with an altered metabolite production

ABSTRACT

A recombinant microbial cell comprises at least one increased expressible enzyme activity controlling anabolic metabolism of ammonia as a nutrient source, the increased enzyme activity being an NADH-dependent activity catalysing the reaction
         (a) 2-oxoglutarate+NH 3 +NADH→glutamate+NAD or being an activity catalysing the reaction   (b) 2-oxoglutarate+glutamine+NADH→2 glutamate+NAD or being an activity catalysing the reaction   (c) glutamate+NH 3 +ATP→glutamine+ADP+Pi.
 
The increased enzyme activity is encoded by a nucleic acid coding sequence linked to an expression signal not natively associated with the nucleic acid coding sequence and is increased as compared to the expression of the enzyme activity when the nucleic acid coding sequence is associated with its native expression signal.

The present application is the national stage under 35 U.S.C. 371 ofPCT/DK99/00397, filed 12 Jul. 1999.

TECHNICAL FIELD OF THE INVENTION

The invention is in the areas of microbial biotechnology, metabolic andgenetic engineering. It relates to a microbial cell wherein theexpression of a number of expressible enzyme activities have been eitherincreased or decreased or eliminated in order to alter the rate ofproduction and/or the yield of a cellular metabolite such as anintermediate product or an end product of a metabolic pathway.

At least one, preferably two or more, of said expressible enzymeactivities, when expressed, mediates a reaction involved in theassimilation of a nutrient source in said cell. Assimilation of anutrient source may involve uptake of said source into the cell and/orconversion of said source within said cell, preferably the incorporationof said source into a biosynthetic product of said cell, saidincorporation being controlled by the metabolic potential and capabilityof said cell.

The invention described herein below relates to a manipulation of aprocess of assimilation of a nutrient source, preferably ammonia, andthe resulting effect thereof on the capability of a microbial cell toproduce one or more primary and/or secondary metabolites such asintermediate products and/or end products of one or more metabolicpathways present in said microbial cell.

BACKGROUND OF THE INVENTION

A living cell carries out a complex network of more than a thousanddifferent reactions simultaneously, with each sequence of reactionsbeing strictly and sensitively controlled in a number of ways so thatundesirable accumulations or deficiencies of intermediates and/or endproducts are normally prevented from occurring. As a result of thisstrict and sensitive control, reactions of great mechanistic complexityand stereochemical selectivity may proceed smoothly under normalphysiological conditions such as ambient pressure, moderate temperatureand a pH near neutrality.

In order to appreciate the complexity and selectivity of the control ofmetabolic networks, it is necessary first to consider specific reactionsequences such as metabolic pathways; the relationship between eachpathway and the cellular architecture; the biological importance of eachmetabolic pathway; and the sensitive and efficient control mechanismsregulating intracellular reaction rates. The totality of intracellularreaction rates is also known as metabolic flux.

The person skilled in the art will be aware of many microbial primaryand secondary metabolites and he will have access to relevant referencecollections on the subject such as the authoritative Bergeys Manual. Theperson skilled in the art will also have to his disposal generalbiochemistry textbooks comprising state of the art insights into thecomplex world of cellular metabolism and biochemistry.

Metabolic engineering may be perceived as a purposeful redesigning ofmetabolic networks generating a change in and/or a redirection of theaerobic and/or the anaerobic metabolism of a microbial cell. State ofthe art metabolic engineering techniques have been described by amongothers Cameron and Chaplen (1997) in Curr. Opin. Biotechnol., vol. 8,pages 175–180, Hahn-Hägerdal et al. (1996) in Ann. New York Acad. Sci.,vol. 782, pages 286–296, Stephanopoulos (1994) in Curr. Opin.Biotechnol., vol. 5, pages 196–200. Stephanopoulos and Sinskey (1993) inTrends Biotechnol., vol. 11, pages 392–396, and Cameron and Tong (1993)in Appl. Biochem. Biotechnol., vol. 38, pages 105–140.

Some microbial cells are potentially recognisable by a singlecharacteristic trait in the form of e.g. a metabolic end productpredominantly produced under a given set of growth conditions.Accordingly, a yeast may well be initially characterised by a productionof ethanol in much the same way as a lactic acid bacterial cell may bepotentially identifiable by a production of lactic acid. However, thecomplex environment wherein microbial cell metabolites are producedevidently leads not only to the formation of a single althoughpredominant metabolite, but rather to a complex set of metabolicintermediates and end products. Many aspects of microbial metabolism andthe regulation thereof are still far from being thoroughly understood.

Cellular metabolism comprises catabolism. i.e. those processes relatedto a degradation of complex macromolecular substances, and anabolism, orthose processes concerned primarily with the synthesis of often quitecomplex organic molecules. Both catabolic and anabolic pathways can beperceived to occur in several stages of complexity—one being aninterconversion of polymers and complex lipids with monomericintermediates; another an interconversion of monomeric sugars, aminoacids, and lipids with relatively simple organic compounds; and yetanother stage being the ultimate degradation to, or synthesis from,inorganic compounds such as CO₂, H₂O, and NH₃.

Catabolic and anabolic metabolism can be further divided into an aerobicand an anaerobic metabolism. i.e. metabolism occurring either in thepresence or absence of oxygen. Many microorganisms are capable ofgrowing in both the presence and absence of oxygen. Some microbial cellsare strictly aerobic and depend absolutely upon an oxidative form ofmetabolism known as respiration, i.e. the coupling of energy generationto an oxidation of nutrients by oxygen.

The conversion of glucose to pyruvate in a cell undergoing activerespiration, i.e. an oxidative breakdown and generation of energy fromnutrient sources by means of a reaction with oxygen, results in theformation of a coenzyme in a reduced form known as nicotinamide adeninedinucleotide, or NADH. NADH is reoxidised through the mitochondrialelectron transport chain in a process that generates additional energyand results in an ultimate transfer of electrons to oxygen.

The coenzyme nicotinamide adenine dinucleotide in its oxidised form(NAD⁺) contains a nicotinamide ring structure that is readily reducibleand thus serves as an oxidising agent. Accordingly, nicotinamide adeninedinucleotide may consist in either a reduced form, NADH, or an oxidisedform, NAD⁺. Many dehydrogenase enzymes, such as alcohol dehydrogenases,have a strong affinity for the oxidised form, NAD⁺. After oxidation of asubstrate, the reduced form of the coenzyme. NADH, leaves the enzyme andis reoxidised by available electron-acceptor systems in the cell. TheNAD⁺ so formed can now bind to another enzyme molecule and repeat thecycle. NAD⁺ and NADH differ from most substrates in that they arecontinually recycled.

By contrast to the oxidative metabolism of the respiratory chain, manymicroorganisms either can or must grow in anaerobic environments whilederiving their metabolic energy from processes that do not involveoxygen. Most of such anaerobically growing microbial organisms derivetheir energy from fermentations characterised by energy-yieldingcatabolic pathways such as glycolysis, wherein a conversion of glucoseresults in formation of products such as e.g. ethanol and CO₂.

Cellular metabolism evidently requires and generates energy, andenergy-yielding metabolic pathways generate many intermediates used innumerous biosynthetic pathways. Cells mostly obtain free energy releasedduring catabolism in the form of ATP. The chemical energy stored as ATPmay be converted to other forms of energy in a process known as energytransduction.

Glycolysis is a major catabolic pathway for degradation of carbohydratesin both aerobically and anaerobically growing microbial cells. The majorinput to glycolysis is glucose and the pathway, comprising a total of 10different reactions, leads to the conversion of one molecule of glucoseto two molecules of pyruvate, with the concomitant generation of ATP aswell as the coenzyme NADH.

The sequence of reactions between glucose and pyruvate can be consideredas two distinct phases, one comprising the first five reactions andconstituting an energy input phase, in which sugar phosphates aresynthesised at the expense of a conversion of ATP to a less energy richmolecule in the form of ADP, and one phase comprising the last fivereactions and representing an energy output phase, in which a transferof a phosphate group to ADP leads to regeneration of ATP. The glycolyticconversion of glucose to pyruvate also involves the concomitantreduction of two moles of NAD⁺ to its reduced equivalent NADH.

Anaerobically growing microbial cells may reduce pyruvate produced bymeans of glycolysis to a variety of metabolic end products such as e.g.ethanol, lactic acid, acetic acid and carbon dioxide. Ethanol productionthrough anaerobic fermentation of a carbon source by the yeastSaccharomyces cerevisiae is one of the best known biotechnologicalprocesses and accounts for a world production of approximately 30billion liters per year. The ethanol yield is lower than a maximum,theoretical yield due to a formation of a number of additional productsaffecting the ethanol yield, such as e.g. biomass, acetate, pyruvate,succinate and glycerol. A de novo synthesis of the first four componentsresults in a net formation of NADH, while a synthesis of glycerol occursunder simultaneous NADH consumption. As ethanol is synthesised without anet formation or consumption of NADH, glycerol formation plays animportant physiological role under anaerobic growth. Glycerol formationleads to a reoxidation of NADH to NAD⁺ and thereby substitutes the roleof oxygen as an electron acceptor.

It is known that in anaerobic cultivations of Saccharomyces cerevisiaeCBS8066, approximately 10% of the carbon source is directed towards theformation of glycerol (Nissen et al., 1997: Verduyn et al., 1990). Aredirection of this amount of carbon towards ethanol production isclearly desirable and would presuppose a reduction in the net formationof NADH in the synthesis of biomass and organic acids.

Accordingly, for the glycolytic pathway to operate anaerobically, i.e.in the absence of oxygen. NADH must be reoxidised to NAD⁺ by means of atransfer of electrons to a suitable electron acceptor so that a steadymetabolic flux can be maintained. Microbial cells growing anaerobicallyhave different ways of transferring such electrons. A simple route usedby lactic acid bacteria consists of simply using NADH to reduce pyruvateto lactate, via the enzyme lactate dehydrogenase. NADH is reoxidised inthe process:Pyruvate+NADH

Lactate+NAD⁻The lactic acid fermentation. i.e. conversion of glucose to lactic acid,is important in the manufacture of cheese. Another importantfermentation involves a conversion of pyruvate to acetaldehyde and CO₂and a reduction of acetaldehyde to ethanol by alcohol dehydrogenase:Acetaldehyde+NADH

Ethanol+NAD⁺When carried out by yeast cells, this fermentation generates the alcoholin alcoholic beverages. Yeast cells used in baking also carry out thisform of fermentation and the CO, produced by pyruvate decarboxylationcauses bread to rise while the ethanol produced evaporates duringbaking. Among many other useful fermentations are those leading to e.g.acetic acid in the manufacture of vinegar and propionic acid in themanufacture of Swiss cheese.

Glycerol formation in cellular metabolism has at least twophysiologically important roles in Saccharomyces cerevisiae—it isinvolved in NADH reoxidation and it acts as an efficient osmolyt thatprotects the cell against lysis under stress conditions.

Synthesis of biomass and organic acids, i.e. succinic acid, acetic acidand pyruvac acid, results in a net formation of intracellular NADH(Oura, 1977; van Dijken & Scheffers, 1986; Nissen et al. 1997). This hasto be balanced by a mechanism in which NADH is reoxidised to NAD⁺ inorder to avoid depletion of the NADH pool. Under anaerobic conditions.NADH reoxidation is not possible by means of the respiratory chain,which is not functioning under such conditions. Instead. NADH isreoxidised to NAD⁺ via formation of glycerol, since synthesis of onemolecule of glycerol from glucose leads to reoxidation of one moleculeof NADH.

Glycerol is also formed and accumulated inside the cell during growthunder osmotic stress conditions and acts as an efficient osmolyt thatprotects the cell against lysis (Ansell et al., 1997; Larsson et al.(1993)). The formation of glycerol occurs via a two step reaction fromdihydroxyacetone phosphate (DHAP) that is catalysed by glycerol3-phosphate dehydrogenase and glycerol 3-phosphate phosphatase,respectively.DHAP+NADH→Glycerol-3-Phosphate+NAD⁺Glycerol-3-Phosphate→Glycerol+PhosphateIn order to be able to produce any metabolic product, the microbial cellneeds an input in the form of both energy and readily assimilablenutrient sources. The metabolism of a microbial cell very muchdetermines the capability of said cell to exploit nutrient sourcespresent in an external environment. Consequently, the metabolism of amicrobial cell is dynamic and the sensitive regulation, direction andredirection of said metabolism is indicative of the responses of saidcell to changing environmental conditions.

Assimilable nutrients such as various sources of nitrogen, carbon,sulphur and phosphor exist in many different forms. Some of these formsmay be readily assimilated by a microbial cell while others cannot beassimilated. In the case of nitrogen, it is essential that a microbialcell is capable of assimilating this nutrient source, as nitrogen formspart of among others i) amino acids in proteins, ii) nucleotides in DNAand RNA, iii) amino sugars in complex polysaccharides, and iv)heterocyclic compounds in various coenzymes.

As described above, catabolic and anabolic pathways occur in differentstages of complexity and one of said stages involves the ultimatedegradation to, or synthesis from, inorganic compounds such as CO₂, H₂O,and NH₃. The majority if not all microbial cells are capable ofassimilating ammonia and converting this source of nitrogen into organicnitrogen compounds—i.e. any organic compound comprising a C—N bond.Ammonia is thus a central metabolite and actually serves as a substratefor no less than five different enzymes that convert it into variousorganic nitrogen-comprising compounds. At physiological pH, the dominantionic species is an ammonium ion, but all of said five reactions involvethe unshared electron pair of NH₃, which is therefore generallyconsidered the reactive species.

Accordingly, microbial cells assimilate ammonia via reactions leading tothe formation of either glutamate, glutamine, asparagine, or carbamoylphosphate. Because carbamoyl phosphate is used only in the biosynthesisof arginine, urea, and the pyrimidine nucleotides, most of the nitrogenending up in amino acids and other nitrogen comprising organic compoundsis assimilated via the two amino acids glutamate and glutamine. Theenzymes responsible for ammonia assimilation in a microbial cell arebriefly introduced herein below.

Glutamate dehydrogenase catalyses the reductive amination of2-oxoglutarate:2-Oxoglutarate+NH₃+NAD(P)H

Glutamate+NAD(P)Microbial cells growing with ammonia as their sole nitrogen source usethe above reaction as a primary route for nitrogen assimilation.

Most microbial cells contain an NADPH-specific form of the glutamatedehydrogenase enzyme, as indicated above, which acts primarily in thedirection of glutamate formation. Interestingly, yeast contain both aNADH-specific form and a NADPH-specific form of the enzyme, each formbeing appropriately regulated, with one form, NADPH, primarily involvedin nitrogen assimilation and the other, NADH, functioning primarily incatabolic metabolism.

The major source of electrons for reductive biosynthesis is NADPH,nicotinamide adenine dinucleotide phosphate. NADP⁺ and NADPH areidentical to NAD⁺ and NADH, respectively, except that the form has anadditional phosphate esterified at C-2′ on the adenylate moiety. NAD⁺and NADP⁺ are equivalent in their thermodynamic tendency to acceptelectrons and they have similar standard reduction potentials. Forreasons not known, nicotinamide nucleotide-linked enzymes that act incatabolic metabolism usually use the NAD⁺/NADH coenzyme pair, whereasthose acting in anabolic pathways tend to use NADP⁺/NADPH.

Glutamate synthase is an enzyme functionally related to glutamatedehydrogenase and catalyses a reaction comparable to that catalysed byglutamate dehydrogenase. However, glutamate synthase functions primarilyin glutamate biosynthesis:2-Oxoglutarate+glutamine+NADPH→2 glutamate+NADP⁺When formed by the action of glutamate dehydrogenase, glutamate canaccept a second ammonia moiety to form glutamine in a reaction catalysedby the enzyme glutamine synthetase:Glutamate+NH₃+ATP→Glutamine+ADP+Pi

This enzyme is named a synthetase, rather than a synthase, because thereaction couples bond formation with the energy released from ATPhydrolysis. However, both enzymes are classified as ligases, but asynthase enzyme does not require ATP.

Glutamine synthetase of E. coli is a dodecamer, whose 12 identicalsubunits form two facing hexagonal arrays. The holoenzyme has amolecular weight of about 600.000. Each catalytic site is formed at aninterface between polypeptide subunits within a hexamer and is made upof residues from two adjacent subunits.

Glutamine occupies a central role in the nitrogen metabolism of anymicrobial cell. The amide nitrogen is used in biosynthesis of severalamino acids, including glutamate, tryptophan, and histidine, purine andpyrimidine nucleotides, and amino sugars. As revealed primarily based onstudies in E. coli, several remarkable and quite extraordinary controlmechanisms for glutamine synthetase mediated reactions interact with oneanother in very complex ways. The activity of glutamine synthetase iscontrolled by two distinct but mutually related mechanisms: Alostericregulation by cumulative feedback inhibition and covalent modificationof the enzyme mediated by a regulatory cascade.

Cumulative feedback inhibition involves the action of no less than eightspecific feedback inhibitors. Those eight inhibitors are eithermetabolic end products of glutamine metabolism (tryptophan, histidine,glucosamine-6-phosphate, carbamoyl phosphate, CTP, and AMP), or they areindicators in various ways of the general status of amino acidmetabolism (alanine, glycine). Quite remarkably, each 50,000-daltonsubunit of glutamine synthetase contains binding sites for each of theeight inhibitors, as well as binding sites for substrates and products.

Each of the eight compounds alone gives only partial inhibition, but incombination the degree of inhibition is increased until a mixture of alleight provides a virtually complete blockage. This ensures that anaccumulation of an end product of one pathway does not shut off thesupply of glutamine needed for another pathway. Glutamine synthetase isalso regulated by means of adenylylation. An enzyme molecule with all 12sites adenylylated is completely inactive, whereas partial adenylylationyields a correspondingly partial inactivation.

Adenylylation and deadenylylation of glutamine synthetase involve acomplex series of regulatory cascades. These regulatory cascades providea responsive mechanism ensuring that, when the supply of activatednitrogen in the form of glutamine is sufficiently high, its furtherbiosynthesis is shut down. In contrast, when activated nitrogen in theform of glutamine is low, 2-oxoglutarate accumulates and, provided thatATP is also abundant, stimulates the activity of glutamine synthetase bythe converse mechanism.

An enzyme comparable to glutamine synthetase, asparagine synthetase,accounts for a significantly smaller amount of ammonia assimilation.Asparagine synthetase uses ammonia or glutamine in catalysing theconversion of aspartate to asparagine. The enzyme cleaves ATPdifferently from the way ATP is cleaved by glutamine synthetase.Asparagine synthetase also differs from glutamine synthetase in thatglutamine is strongly preferred as a substrate over ammonia.

Carbamoyl phosphate synthetase is another enzyme involved in theassimilation of ammonia in microbial cells. Ammonia or glutamine mayboth serve as the nitrogen donor.NH₃+HCO₃ ⁻+2 ATP→carbamoyl phosphate+2ADP+P_(i)Glutamine+HCO₃ ⁻+2ATP+H₂O→Carbamoyl phosphate+2ADP+P_(i)+glutamate

The bacterial enzyme catalyses both reactions, although glutamine is apreferred substrate. Eukaryotic microbial cells contain two forms of theenzyme. Form I is located in the mitochondria and has a preference forammonia as substrate, whereas form II is present in the cytosol and hasa strong preference for glutamine.

Several examples of metabolically engineered microorganisms aredescribed in the patent literature. EP 0 733 712 A1 discloses a methodof production of preferably an amino acid by culturing a metabolicallyengineered microbial cell, preferably an Escherichia coli cell, with asupposedly increased expression or productivity of NADPH and isolatingsaid amino acid.

WO 96/41888 discloses a yeast cell having a modified alcohol sugarfermentation due to an altered expression of a gene encoding a NADHdependent glycerol-3-phosphate dehydrogenase activity.

EP 0 785 275 A2 discloses a yeast comprising constitutive expression ofa gene encoding an enzyme activity involved in hexose transport.

EP 0 645 094 A1 discloses the use of a yeast comprising a glycolyticpathway comprising a futile cycle generated by means of a constitutiveexpression of genes encoding fructose-1,6-biphosphatase andphosphoenolpyruvate carboxykinase.

U.S. Pat. No. 5,545,556 discloses a yeast strain having a reduced orincreased production of glycerol mediated by mutations in variousgene-encoded products.

None of the above disclose a microbial cell wherein the expression of anumber of expressible enzyme activities involved in nutrientassimilation are either increased or decreased or eliminated in order toalter the rate of production and/or the yield of a cellular metabolitesuch as an intermediate product or an end product of a metabolicpathway.

SUMMARY OF THE INVENTION

It has now surprisingly been discovered that it is possible to operablylink the process of nutrient assimilation in a microbial cell such ase.g. a yeast cell with an increased production of a metabolite like e.g.ethanol. The invention is based on the unexpected and surprising findingthat it is possible to operably link an energy requiring reaction suchas assimilation of a nutrient source with an energy yielding reactionsuch as the formation of a desirable metabolic product like e.g.ethanol.

When the nutrient source is a nitrogen source such as ammonia or anitrogen source convertible into ammonia, it is preferred tosubstantially reduce or eliminate the expression in a microbial cell ofan enzyme, glutamate dehydrogenase, which, under normal physiologicalconditions, is the predominant enzyme activity involved in ammoniaassimilation, and concomitantly with said reduction or elimination,increase the expression of one or both of two additional enzymeactivities, glutamate synthase and glutamine synthetase, both of whichare capable of assimilating ammonia into glutamate under the consumptionof ATP and re-oxidation of NADH. Accordingly, overexpression ofglutamate synthase and glutamine synthetase in a microbial cell,preferably a yeast cell, under anaerobic conditions, generates areduction of intracellular ATP levels and potentially a depletion of thepool of ATP available to the cell. Under anaerobic conditions, the cellis capable of counteracting the reduction in the ATP pool by producingan increased amount of a metabolite such as e.g. ethanol via an ATPyielding reaction.

The world ethanol production reached an estimated 31.3 billion liters in1996. Approximately 80% were produced by anaerobic fermentation ofvarious sugar sources by Saccharomyces cerevisiae. Accordingly, ethanolis one of the most important biotechnological products with respect toboth value and amount. Two thirds of the production is located in Braziland in the United States with the primary objective of using ethanol asa renewable source of fuel. The demand and growth of this market isexpected to give rise to a substantial growth in the ethanol productionindustry in the future. Hence, there are strong economic incentives tofurther improve the ethanol production process.

The price of the sugar source is a very important process parameter indetermining the overall economy of ethanol production. Hence, it is ofgreat interest to optimise the ethanol yield in order to ensure anefficient utilisation of the carbon source. Besides biomass and carbondioxide, a number of by-products are formed during an anaerobicfermentation of Saccharomyces cerevisiae (Oura, 1977). Glycerol is themost dominant of these compounds, consuming up to 4% of the carbonsource in industrial fermentations. Accordingly, it is highly desirableto eliminate formation of this compound, when it is not wanted, andredirect the metabolic flux towards ethanol production. If successfullyachieved, it should in theory be possible to increase the ethanol yieldby a maximum of 4%, corresponding to an increase in the world productionof ethanol of 1.25 billion liters per year without any additional costs.

Although it is extremely difficult to alter or redirect a microbialmetabolism, such as an anaerobic yeast metabolism, it may never the lessbe desirable to alter a “traditional” profile of primary and/orsecondary metabolites in order to achieve a different composition or“product mix”, or in order to increase or decrease or even eliminate theproduction of some metabolites present in said profile.

The surprising and unexpected finding described in the present inventionmakes it possible to i) manipulate the process of assimilation of anutrient source such as ammonia into or in a microbial cell and ii)correlate said manipulation of said process with a provision of analtered profile of produced metabolites including e.g. ethanol andglycerol.

The correlation of assimilation of a nutrient source with primary and/orsecondary metabolite production is achieved by bridging saidassimilation with an altered production of a metabolite—an intermediateproduct or an end product of a metabolic pathway—through an extremelycomplex and not thoroughly understood network of metabolic reactionsguiding the flux of metabolites in a microbial cell.

Accordingly, in a first aspect of the invention there is provided amicrobial cell comprising

-   -   i) a first expressible enzyme activity which, when expressed in        said microbial cell, is controlling assimilation in said cell of        a nutrient source, said expression of said first enzyme activity        in said microbial cell being either novel or altered as compared        to the expression of said first enzyme activity in a comparable        wild-type microbial cell or a comparable isolated microbial        cell, and optionally    -   ii) a second expressible enzyme activity which, when expressed        in said microbial cell, is controlling assimilation in said cell        of a nutrient source, said expression of said second enzyme        activity in said microbial cell being either novel or altered as        compared to the expression of said second enzyme activity in a        comparable wild-type microbial cell or a comparable isolated        microbial cell, said second expressible enzyme activity being        non-identical to said first expressible enzyme activity, and    -   iii) a reduced expression or no expression of a third        expressible enzyme activity which, when expressed in said        microbial cell, is controlling assimilation in said cell of a        nutrient source, said third expressible enzyme activity being        non-identical to any and both of said first and second        expressible enzyme activities, and further optionally    -   iv) a fourth expressible enzyme activity which, when expressed        in said microbial cell, is controlling an intracellular redox        system of said cell, said expression of said fourth enzyme        activity in said microbial cell being either novel or altered as        compared to the expression of said fourth enzyme activity in a        comparable wild-type microbial cell or a comparable isolated        microbial cell, said fourth expressible enzyme activity being        non-identical to each and all of said first, second and third        expressible enzyme activities.

In another aspect of the invention, the microbial cell forms part of acomposition further comprising a carrier. In yet another aspect there isprovided a novel nucleotide sequence encoding an expressibletranshydrogenase enzyme activity capable of controlling an intracellularredox system of a microbial cell. There is also provided a recombinantDNA-replicon in the form of a vector comprising said nucleotide sequenceencoding said expressible transhydrogenase enzyme, and a microbial cellharbouring said nucleotide sequence or said vector. The invention alsopertains to an amino acid sequence encoded by said nucleotide sequence.

In a further aspect, the invention is related to a microbial cell or acomposition for use in the production of a first or a second metabolite.There is also provided a microbial cell or a composition for use in apreparation of a drinkable or an edible product. In another aspect thereis provided a microbial cell or a composition for use in a production ofa first or second metabolite for use in a drinkable or an edibleproduct.

In a yet further aspect there is provided the use of a microbial cell ora composition in a production of a first or a second metabolite. Theinvention also pertains to the use of a microbial cell or a compositionin the production of a first or second metabolite for use in a drinkableor an edible product.

In an even further aspect there is provided a method of producing afirst metabolite, said method comprising the steps of

-   -   i) cultivating a microbial cell or a composition comprising said        cell in a suitable growth medium and under such conditions that        said microbial cell is producing said first metabolite, and        optionally    -   ii) isolating said first metabolite in a suitable form, and        further optionally    -   iii) further purifying said isolated first metabolite.

In an even further aspect there is provided a method of constructing amicrobial cell according to the invention, said method comprising thesteps of

-   -   i) operably linking a nucleotide sequence encoding said first        expressible enzyme activity with an expression signal not        natively associated with said nucleotide sequence, and/or    -   ii) operably linking a nucleotide sequence encoding said second        expressible enzyme activity with an expression signal not        natively associated with said nucleotide sequence, and    -   iii) eliminating said third expressible enzyme activity from        said microbial cell or optionally operably linking a nucleotide        sequence encoding said third expressible enzyme activity with an        expression signal not natively associated with said nucleotide        sequence, said expression signal generating a reduced expression        of said nucleotide sequence, and    -   iv) introducing said operably linked nucleotide sequences        obtained under i) and iii), and optionally the nucleotide        sequence obtained under ii), into said microbial cell, or    -   v) introducing said operably linked nucleotide sequence obtained        under i), and optionally the nucleotide sequence obtained under        ii), into said microbial cell obtained under iii) wherein said        third expressible enzyme activity has been eliminated.

Preferred embodiments of the above-mentioned aspects of the inventionare described herein below.

DETAILED DESCRIPTION OF THE INVENTION

Attempts have been made to increase ethanol formation in yeast byelimination of glycerol synthesis through deletions of GPD1 and GPD2,encoding the two existing isoenzymes of glycerol 3-phosphatedehydrogenase (Björkqvist et al. 1997). The double deletion mutant isunable to grow under anaerobic conditions due to accumulation ofintracellular NADH. NADH is accumulated since no alternative pathways toreoxidise NADH under these growth conditions exist in S. cerevisiae.Elimination of the capability of generating glycerol results in a strainwith a high sensitivity to osmotic stress. Osmotic stress is caused bygrowth of a cell in an industrial growth medium high in concentrationsof various carbon sources and salts. Deletion of one of the genesresults neither in a significant reduction in glycerol formation nor inan increased ethanol formation (Liden et al., 1997: Nissen et al.,1998a,b).

Consequently, metabolic engineering of the synthesis of glycerol has sofar not proved successful and no significant increase in ethanolproduction in a metabolically engineered S. cerevisiae strain has beenreported.

It has now surprisingly been found that it is possible to implement astrategy comprising a reduction of a surplus of NADH formed by catabolicmetabolism concomitantly with an increased consumption of ATP in thesynthesis of biomass.

Ammonia is often used as a nitrogen source in industrial fermentationsof S. cerevisiae. Following transport across the membrane into thecytoplasm, ammonia or the ammonium ion is converted into glutamate byassimilation with 2-oxoglutarate. In wild-type cells this reaction iscatalysed by an NADPH-dependent glutamate dehydrogenase encoded by GDH1(Moye et al., 1985):2-Oxoglutarate+NH₄ ⁺+NADPH→Glutamate+NADP⁺

Two other glutamate dehydrogenases, encoded by GDH2 and GDH3, are alsopresent in S. cerevisiae. Gdh2p normally catalyses the opposite reactionas that of Gdh1p under formation of NADH (Miller and Magasanik, 1990;Miller and Magasanik, 1991; Coschigano et al., 1991: Courchesne andMagasanik, 1988). This reaction occurs when nitrogen sources other thanammonium and glutamine are used. The reaction may also play a role inammination reactions during synthesis of various amino acids.

The activity of Gdh2p is 70 times lower than the activity of Gdh1p, whenammonium is used as nitrogen source (Nissen et al. 1997). It has beendemonstrated, as described herein below and in Example 1, that formationof glycerol can be reduced by deleting GDH1 and overexpressing GDH2.These manipulations result in a genetically modified yeast strainsynthesising glutamate under consumption of NADH rather than NADPH(Nissen et al., 1998a). However, this reduction in glycerol formationdid not result in an overall increase in the ethanol yield. The functionof NADPH-dependent Gdh3p is unknown. No activity of a NADPH-dependentglutamate dehydrogenase can be measured in cells with a deletion in GDH1when ammonium is used as nitrogen source, suggestion an involvement ofthis enzyme primarily when other nitrogen sources are used (Nissen etal. 1998a; Avendan{hacek over (o)} et al., 1997).

Interestingly, there exist another system capable of synthesisingglutamate in S. cerevisiae. This system consists of two coupledreactions, catalysed by glutamate synthase, encoded by GLT1, andglutamine synthetase, encoded by GLN1 (Cogoni et al. 1995, Miller andMagasanik, 1983). The reactions mediated by said enzymes are brieflyillustrated below.Glt1p: 2-Oxoglutarate+Glutamine+NADH→2 Glutamate+NAD⁺Gln1p: Glutamate+NH₄ ⁺+ATP→Glutamine+ADP+P_(i)Glt1p+Gln1p: 2-Oxoglutarate+NH₄ ⁺ NADH+ATP→Glutamate+NAD⁺+ADP+P_(i)

In wild-type cells, the activity of Glt1p is 80 times lower than theactivity of Gdh1p. Hence, the system can be expected to have a verylimited effect on the assimilation of ammonium in a yeast cell.

If GDH1 is deleted from a yeast cell, the cell becomes unable toassimilate ammonium by the means of NADPH-dependent glutamatedehydrogenase activity. This leads to a decrease in the maximum specificgrowth rate to half of that observed for a wild-type yeast cell (Nissenet al. 1998a), this is probably due to a limitation in glutamatesynthesis, since other enzymes potentially involved in this process areexpressed at very low levels, as described herein above.

A double mutant in GDH1 and GLT1 is unable to grow on ammonium asnitrogen source, indicating that glutamate synthase potentially couldhave a role as a backup system in the synthesis of glutamate (Miller andMagasanik, 1990). Thus, it was hypothesised that by overexpressing GLT1and GLN1 in a Δgdh1 mutant, it should be possible to alleviate and/oreliminate the limiting effect of glutamate synthesis on μ_(max) andobtain a S. cerevisiae strain with a reduced surplus formation of NADHand an increased consumption of ATP in biomass synthesis.

It has been demonstrated that deletion of GDH1, encoding theNADPH-dependent glutamate dehydrogenase 1, in a wild-type strain of S.cerevisiae resulted in an increase in ethanol formation and a decreasein glycerol formation (Nissen et al. 1998a,b). Unfortunately, theethanol productivity was significantly affected by the deletion sincethe maximum specific growth rate of the Δgdh1 mutant was approximatelyhalf of that of the wild-type strain.

The present invention has demonstrated for the first time thatoverexpression of GLN1, encoding glutamine synthetase, and GLT1,encoding glutamate synthase, in a Δgdh1 mutant results in a significantincrease in the maximum specific growth rate, as compared to the Δgdh1mutant, as well as a substantially increased yield of ethanol.

Consequently, a novel pathway for glutamate synthesis has been shown tosubstitute the role of an NADPH-dependent glutamate dehydrogenase in theassimilation of ammonium and 2-oxoglutarate to glutamate.

The maximum specific growth rate of a strain according to the invention,TN19, was 90% of the maximum specific growth rate of the wild-type andthis clearly limits the increase in the specific ethanol productivitythat was obtained. This problem can most likely be solved by increasingthe specific activities of Gln1p and Glt1p even more. In one embodimentof the invention, a five-fold increase in the specific activity ofglutamate synthase was obtained by insertion of the PGK promoter intochromosome IV in front of the structural gene of the enzyme. This levelof activity can be increased significantly by e.g. inserting more copiesof the gene into the chromosome by means of e.g. amplification. It isalso possible to subject the expression system in question to a detailedgenetic and/or biochemical analysis in order to find ways of increasingthe expression.

In an earlier study, it was demonstrated that overexpression of GDH2,encoding the NADH-dependent glutamate dehydrogenase, in a Δgdh1 mutantled to an increase in the maximum specific growth rate from 0.22 h⁻¹ to0.39 h⁻¹ (Nissen et al. 1998a). The specific activity of Gdh2p in thisstrain was 0.625 units per mg TCP, ten-fold higher than in thewild-type, which illustrates that the activity of Glt1p and Gln1p shouldbe increased further in order to achieve the same maximum specificgrowth rate as the wild-type.

The limited increase in the fermentation time of TN19 compared to TN1might not be a serious problem, as it is the substrate cost that is aprimary factor determining the overall economy of ethanol production.This means that the observed increase in the ethanol yield of TN19,combined with the relative small reduction of the maximum specificgrowth rate, represents a breakthrough in and a valuable contribution toan optimisation of microbial ethanol production.

Furthermore, the results obtained in this study showed that even thoughit is very difficult to perform a metabolic engineering process, theproposed strategy of increasing the ethanol yield in S. cerevisiae bymetabolic engineering of pathways involved in nutrient assimilation andbiomass synthesis is a major success.

Accordingly, it has been demonstrated that a mere reduction of glycerolformation via metabolic engineering of NADH- and NADPH-consumingreactions does not necessarily result in an increased flux towardsethanol, and said reduced glycerol formation must accordingly, asconvincingly demonstrated herein by the provision of impressive resultsrepresenting a breakthrough in microbial ethanol production, be combinedwith an increased consumption of ATP in biomass formation in order toredirect carbon flux from glycerol towards an increased production ofethanol.

The microbial cell according to the invention comprises an alteredcomposition of expressible enzyme activities and/or an alteredexpression thereof. In principle, any microbial cell capable of i)assimilating a nutrient source, ii) metabolising said source, and iii)producing a biosynthetic product in the form of e.g. one or more primaryand/or secondary metabolites, forms part of the invention. Theexpressible enzyme activities such as a first and/or second expressibleenzyme activity are preferably operably linked to an expression signalnot natively associated with said activities.

In a preferred embodiment there is provided a microbial cell comprising

-   -   i) an increased expression of said first expressible enzyme        activity controlling assimilation in said cell of a nutrient        source, said first expressible enzyme activity being operably        linked to an expression signal not natively associated with said        first enzyme activity, and optionally    -   ii) an increased expression of said second expressible enzyme        controlling assimilation in said cell of a nutrient source, said        second expressible enzyme activity being operably linked to an        expression signal not natively associated with said second        enzyme activity, and    -   iii) a reduced expression or no expression of said third        expressible enzyme activity which, when expressed in said        microbial cell, is controlling assimilation in said cell of a        nutrient source, said third expressible enzyme activity being        optionally operably linked to an expression signal not natively        associated with said third enzyme activity, and further        optionally    -   iv) a fourth expressible enzyme controlling an intracellular        redox system of said cell, said fourth expressible enzyme        activity being operably linked to an expression signal not        natively associated with said fourth enzyme activity.

In another preferred embodiment, the microbial cell of the inventioncomprises a further expressible enzyme activity, said furtherexpressible enzyme activity, when expressed, mediates an energy yieldingfirst reaction resulting in a production of a first metabolite saidfirst reaction being operably linked to an energy requiring secondreaction resulting in assimilation of a nutrient source. The microbialcell is preferably one wherein said energy requiring second reactionresulting in assimilation of a nutrient source is controlled at least bysaid first and/or second expressible enzyme activity.

In yet another embodiment there is provided a microbial cell comprisinga further expressible enzyme activity, said further expressible enzymeactivity, when expressed, mediates an energy yielding first reactionresulting in a production of a first metabolite, said furtherexpressible enzyme activity, when expressed at an increased level,results in an increased production of said first metabolite, saidincreased expression of said further expressible enzyme activity and/orsaid increased production of said first metabolite is operably linked toan increased expression of said first and/or second expressible enzymeactivity.

The expression of said further expressible enzyme activity preferablyresults in the production of an intermediate or an end product of ametabolic pathway such as metabolites like e.g. lactic acid, aceticacid, propionic acid or ethanol, or a combination thereof. The energyyielding first reaction may accordingly be mediated by a dehydrogenaseenzyme such as e.g. an organic acid dehydrogenase such as a lactatedehydrogenase, or by an alcohol dehydrogenase mediating the formation ofethanol from acetaldehyde.

A primary metabolite is any metabolite forming part of a major metabolicpathway shared by a number of comparable microorganisms such asmicroorganisms within the same species or subspecies. Major metabolicpathways are understood to comprise glycolysis, citric acid cycle,gluconeogenesis, pentose phosphate pathway, urea cycle, and the like.

A secondary metabolite is any organic compound forming part of minorpathways that are “branched off” the above-mentioned major metabolicpathways. The secondary metabolites may well be produced by some memberswithin a species and not by others. An introduction to secondarymetabolites is provided by Herbert (1981) in “The Biosynthesis ofSecondary Metabolites” (Chapman and Hall, London, England).

The microbial cell in question may thus be a microbial eukaryote or amicrobial prokaryote. Among microbial eukaryotes are many yeast andfungal cells preferred, such as yeast cells of the speciesSaccharomyces, Schizosaccharomyces and Pichia, such as e.g.Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris andthe like, as well as algae such as e.g. Chiamydomonas reinhardi, slimemoulds such as e.g. Diclyostelium discoideum and filamentous fungi.Preferred filamentous fungi are species of Neurospora and Aspergillussuch as e.g. Neurospora crassa, Aspergillus nidulans, Aspergillus niger,Aspergillus oryzae and Penicillium chrysogenum. Particularly preferredare also many industrially relevant yeast cells, slime moulds andfilamentous fungi providing a production of products such as e.g.antibiotics, steroids, pigments, enzymes, organic alcohols and acids,amino acids, polysaccharides and the like.

Among preferred microbial prokaryotes are bacterial cells such asGram-positive species such as e.g. Bacillus subtilis, Bacillusthuringensis, Bacillus licheniformis, Bacillus amyloliquefaciens,Bacillus cereus, Bacillus lentus and Bacilus stearothermophilus, speciesof Corynebacterium and Propionibacterium as well as Gram-negativespecies such as Escherichia coli. Particularly preferred are also lacticacid bacterial species such as e.g. Lactococcus lactis, Lactococcuslactis subsp. lactis, Lactococcus lactis subsp cremoris, Lactococcuslactis subsp. diacetylactis, Leuconostoc species, Lactobacillus species,Pediococcus species and similar industrially relevant species like e.g.Bifidobacterium.

The nutrient source is any nutrient source capable of sustainingmicrobial growth by e.g. being assimilated into a biosynthetic productthat can be utilised by a microbial cell or be converted i.e.metabolised into a further biosynthetic product, said utilisation and/ormetabolism involving one or more energy yielding metabolic reactions.Consequently, it will be understood that any nutrient source that isassimilable and metabolisable by a microbial cell forms part of theinvetnion. The step of assimilation shall be understood to comprise bothuptake of said source into said cell as well as conversion of saidsource into a biosynthetic product—an intermediate metabolite—withinsaid cell. In a more narrow scope of the understanding of the term,assimilation shall be meant preferably to comprise the assimilation thattakes place within the cell without necessarily being limited to thisstep of the assimilation process.

In a preferred embodiment of the invention there is provided means foran increased efficiency of an uptake of a nutrient source into amicrobial cell and/or an increased efficiency of assimilation withinsaid cell. The term efficiency shall be understood to comprise, thatboth uptake and/or intracellular conversion takes place at a fasterrate, i.e. increased amounts of nutrients are taken up and/ormetabolised per unit time, or per unit cell time per unit cell mass, insaid cell according to the invention, as compared to a comparablewild-type cell or a comparable isolated cell. The person skilled in theart will be aware as to how a trans-membrane transport and an internalturnover of an assimilated nutrient source may be monitored.

The nutrient source is preferably a nitrogen source, a carbon source, asulphur source, or a phosphor source, more preferably a nitrogen source.Preferred assimilable nitrogen sources comprise ammonia, ammonium ions,nitrite ions, and nitrate ions. The assimilable nitrogen sourceaccording to the invention is more preferably ammonia and ammonium ionsand most preferably ammonia. It will be understood that the inventionpertains to all nitrogen containing nutrient sources capable of beingconverted into ammonia by oxidation including biological oxidation or byreduction including biological reduction. The skilled person will beaware of the fact that biological oxidations and/or reductions may welloccur in the absence of oxygen as a final electron acceptor.

The microbial cell according to the invention comprises a firstexpressible enzyme activity which, when expressed in said microbialcell, is controlling assimilation in said cell of a nutrient source,preferably a nitrogen source such as e.g. ammonia, said expression ofsaid first enzyme activity in said microbial cell being either novel oraltered as compared to the expression of said first enzyme activity in acomparable wild-type microbial cell or a comparable isolated microbialcell.

In a preferred embodiment, the microbial cell comprises a first and asecond expressible enzyme activity which, when expressed in saidmicrobial cell, are controlling assimilation in said cell of a nutrientsource, preferably a nitrogen source such as e.g. ammonia, saidexpression of said first and second enzyme activities in said microbialcell is either novel to said cell or altered as compared to theexpression of said first and second enzyme activities in a comparablewild-type microbial cell or a comparable isolated microbial cell, saidfirst and second expressible enzyme activities being non-identical toone another.

An expressible enzyme activity mediated facilitation of assimilation ofa nutrient source is understood to comprise the capability of saidmicroorganism to carry out a metabolic reaction leading to assimilationin the form of uptake and/or intracellular conversion of said nutrientsource. An intracellular conversion is understood to comprise thesynthesis of a biosynthetic product by fusion of said nutrient source—inthe uptakable form such as a directly assimilable form or in asubsequently processed form—with a metabolite being synthesised by saidcell, said fusion generating a metabolisable biosynthetic product. It isparticularly preferred that the expressible enzyme activity according tothe invention, when expressed, is mediating a biosynthetic reaction.

An altered expression of said first and/or second expressible enzymeactivity in the microbial cell according to the invention shall beunderstood to comprise any expression that differs with respect to therate of product formation or with respect to the amount of productformed as compared to a comparable microbial cell. Accordingly, if awild-type microbial cell is subjected to the metabolic engineeringmanipulations according to the invention, the skilled person willcompare the expression of said first and/or second expressible enzymeactivities provided in the metabolically engineered cell with theexpression of the same activities in the wild-type microbial cell.

Generally, the person skilled in the art will preferably analyse—andcompare with one another—similar or near identical microbial cells suchas identical cells with and without an expressible enzyme activityaccording to the invention. This is standard laboratory practise and theperson skilled in the art will know how to conduct such an analysis sothat it may form a basis for a direct comparison of e.g. an expressedenzyme activity or an expressed coenzyme or an expressed redox systemwithin the meaning of those terms as set out herein below.

Preferably, the person skilled in the art will want to compare microbialcells to cells of at least the same species and more preferably tocompare said cells to cells of at least the same subspecies.

Accordingly, if an isolated microbial cell such as e.g. an industrialstrain or a strain in a culture collection is subjected to the metabolicengineering manipulations according to the invention, the skilled personwill compare the expression of said first and/or second expressibleenzyme activities provided in the metabolically engineered cell with theexpression of the same activities in the industrial strain or themicrobial cell of the culture collection.

The skilled artisan will know how to culture comparable strains such asstrains of the same species or subspecies under identical orsubstantially similar conditions so as to provide a basis for performingthe comparison between the relevant enzyme activities. The personskilled in the art will also know how to perform an enzymatic assay foruse in said comparison and being indicative of the formation of abiosynthetic product resulting from the action of said first and/orsecond expressible activities, when expressed, and he will be aware ofthe potential of transcriptional and/or translational fusions inmonitoring expression of said expressible enzyme activities undercomparable conditions. The skilled person will also be able to performimmunoassays including quantitative immunoprecipitations. An analysis ofgene expression is available in e.g. Old and Primrose (1985): Principlesof Gene Manipulation—An introduction to genetic engineering (Thirdedition). Blackwell Scientific Publications, Oxford, England.

The altered expression of said first and/or second expressible enzymeactivity in the microbial cell according to the invention shallpreferably be understood to comprise an increased expression as comparedto the expression in a comparable microbial cell. Accordingly, any ofsaid first or second expressible enzyme activity, when expressed, is,independently of the other, increased by a factor of at least 1.02, suchas a factor of at least 1.04, for example 1.06, such as 1.08, forexample 11.10, such as at least 1.12, for example 1.14, such as 1.16,for example 1.18, such as 1.2, for example 1.25, such as 1.3, forexample 1.4, such as 1.5, for example 1.6, such as 1.7, for example 1.8,such as 1.9, for example 2.0, such as 2.25, for example 2.5, such as 3,for example 3.5, such as a factor of at least 4, for example 4.5, suchas 5, for example 6, such as 7, for example 8, such as 9, for example10, such as 15, for example 20, such as 25, for example 30, such as 35,for example 40, such as 50, for example 60, such as 80, for example atleast 100, such as 150, for example 200, such as 250, for example 300,such as 350, for example 400, such as 500, for example 600, such as 800,for example at least 1000, such as 1500, for example 2000, such as 2500,for example 3000, such as 3500, for example 4000, such as at least 5000,for example 6000, such as 8000, for example at least 10000, such as15000, for example 20000, such as at least 25000, for example 30000,such as 35000, for example 40000, such as a factor of at least 50000.

However, an altered expression shall not be limited to an increasedexpression. A reduced expression of said expressible activities shouldalso be understood to be comprised by the term altered expression.

A biosynthetic reaction mediated by said first or second expressibleenzyme activity, when expressed, is preferably a reaction capable ofbeing carried out by action of a metabolite synthase enzyme, morepreferably by an allosteric metabolite synthase enzyme, and even morepreferably is said reaction carried out by an expressible enzymeactivity which, when expressed, is exhibited by a glutamate synthase.

In a particularly preferred embodiment of the invention, said glutamatesynthase activity is that of GLT1 of Saccharomyces cerevisiae such ase.g. that of TN17 deposited under DSM Accession Number 12275 or anactivity functionally equivalent therewith. A functionally equivalentactivity is any activity capable of carrying out the same reaction withthe provision of a similar outcome as that resulting from the reactionbeing carried out by the above-mentioned GLT1 encoded polypeptide ofSaccharomyces cerevisiae. When the expressible enzyme activity is anactivity exhibited by a glutamate synthase, the microbial cell ispreferably a yeast cell and more preferably a Saccharomyces cerevisiaecell.

Another biosynthetic reaction mediated by said first or secondexpressible enzyme activity, when expressed, is preferably a reactioncapable of being carried out by action of a metabolite synthetaseenzyme, and more preferably is said reaction carried out by anexpressible enzyme activity which, when expressed, is exhibited by aglutamine synthetase.

It is evident that, as described herein above, an extremely complex andintricate enzyme being regulated as strictly and sensitively asglutamine synthetase does not form an obvious candidate for redirectingthe metabolic flux of biosynthetic reactions related to the assimilationof ammonia in a microbial cell.

In a particularly preferred embodiment of the invention, said glutaminesynthetase activity is that encoded by GLN1 of Saccharomyces cerevisiaesuch as e.g. that of TN15 deposited under DSM Accession Number 12274 oran activity functionally equivalent therewith. A functionally equivalentactivity is any activity capable of carrying out the same reaction withthe provision of a similar outcome as that resulting from the reactionbeing carried out by the above-mentioned GLN1 encoded activity ofSaccharomyces cerevisiae. When the expressible activity is an activityexhibited by a glutamate synthase, the microbial cell is preferably ayeast cell and more preferably a Saccharomyces cerevisiae cell.

Accordingly, in a particularly preferred embodiment there is provided amicrobial cell, preferably a yeast cell, wherein said first expressibleenzyme activity is a metabolite synthase activity, more preferably anallosteric metabolite synthase activity, and even more preferably aglutamate synthase activity and wherein said second expressible enzymeactivity is a metabolite synthetase activity, preferably a glutaminesynthetase activity.

In another preferred embodiment is said first and/or second expressibleenzyme activity a ligase activity or an NADH-dependent glutamatedehydrogenase activity or a NADPH-dependent glutamate dehydrogenaseactivity.

The microbial cell according to the invention may—in addition to anexpressible first and/or second enzyme activity—also comprise a thirdexpressible enzyme activity, said activity, when expressed in saidmicrobial cell, preferably a yeast cell, is controlling assimilation insaid cell of a nutrient source, said expression of said third enzymeactivity in said microbial cell, preferably a yeast cell, being eithernovel or altered as compared to the expression of said third enzymeactivity in a comparable wild-type microbial cell or a comparableisolated microbial cell, said third expressible enzyme activity beingnon-identical to any and both of said first and second expressibleenzyme activities.

There is also provided an embodiment of the invention wherein said thirdexpressible enzyme activity has been deleted from and no longer ispresent in said cell. It is particularly preferred to delete said thirdexpressible enzyme activity when the microbial cell is a yeast cell, butthe activity may also be deleted from any other eukaryotic microbialcell or from a prokaryotic microbial cell.

Reference is made to the above-mentioned comments and argumentsconcerning a definition of terms such as assimilation, nutrient source,altered expression and comparable microbial cell. The interpretationsindicated herein above also apply with respect to said expressible thirdenzyme activity.

Accordingly, in one preferred embodiment according to the invention, thethird expressible enzyme activity is preferably a metabolitedehydrogenase activity, and more preferably a glutamate dehydrogenaseactivity which is either present in said microbial cell and preferablyin a reduced amount, more preferably in a substantially reduced amount,or eliminated from said cell by means of e.g. deletion of a nucleotidesequence encoding said activity or by effectively repressing theexpression of said expressible third enzyme activity.

Consequently, an altered expression in said microbial cell according tothe invention of said third expressible enzyme activity, preferably aglutamate dehydrogenase activity and even more preferably a NADPHdependent glutamate dehydrogenase activity, shall be understood tocomprise a decreased expression as compared to the expression of saidactivity in a comparable microbial cell. Accordingly, the expression ofsaid third expressible enzyme activity, when expressed, is decreased byat least 1 percent, such as decreased by at least 2 percent, for example4 percent, such as 6 percent, for example at least 8 percent, forexample at least 10 percent, such as 12 percent, for example 14 percent,such as 16 percent, such as at least 18 percent, for example at least 20percent, such as 22 percent, for example 24 percent, such as 26 percent,such as at least 28 percent, for example at least 30 percent, such as 32percent, for example 34 percent, such as 36 percent, for example 38percent, such as at least 40 percent, for example 42 percent, such as 44percent, for example 46 percent, such as 48 percent, such as at least 50percent, for example 52 percent, such as 54 percent, for example 56percent, such as 58 percent, such as at least 60 percent, for example 62percent, such as 64 percent, for example 66 percent, such as 68 percent,such as at least 70 percent, for example 72 percent, such as 74 percent,for example 76 percent, such as 78 percent, such as at least 80 percent,for example 82 percent, such as 82 percent, for example 84 percent, suchas 86 percent, such as at least 88 percent, for example 90 percent, suchas 92 percent, for example 94 percent, such as 96 percent, for exampleat least 98 percent, such as 99 percent, for example 99.2 percent, suchas at least 99.4 percent, for example 99.6 percent, such as 99.8percent, for example 99.9 percent, such as 99.92 percent, for example99.94 percent, such as 99.96 percent, for example 99.98 percent, such as99.99 percent, for example decreased to such an extend that saidexpression is unassayable using standard state of the art assays and/orsaid expression is effectively repressed and/or substantiallyeliminated.

However, an altered expression shall not be limited to a decreasedexpression. An increased expression of said third expressible enzymeactivity shall also be understood to be comprised by the term alteredexpression.

In a particularly preferred embodiment of the invention said glutamatedehydrogenase activity is that of a GDH1 encoded polypeptide ofSaccharomyces cerevisiae such as e.g. TN1, or an activity functionallyequivalent therewith. A functionally equivalent activity is any activitycapable of carrying out the same reaction with the provision of asimilar outcome as that resulting from the reaction being carried out bythe above-mentioned GDH1 encoded polypeptide of Saccharomycescerevisiae. When the glutamate dehydrogenase activity is encoded by GDH1of Saccharomyces cerevisiae, the microbial cell is preferably a yeastcell and more preferably a Saccharomyces cerevisiae cell.

Microbial cells pertaining to the invention have been deposited with theDSM under Accession Numbers 12267, 12268, 12274, 12275, 12276 and 12277as Saccharomyces cerevisiae strains TN4, TN9, TN15, TN17, TN19, andTN22, respectively.

Accordingly, said third expressible enzyme activity, preferably in theform of a glutamate dehydrogenase activity and when expressed, is eitherexpressed at a substantially reduced level, not expressed at all in saidcell due to e.g. an efficient repression of expression, or the activityhas been eliminated altogether from said cell. It is particularlypreferred that a DNA sequence encoding said third expressible enzymeactivity, preferably a glutamate dehydrogenase activity, and/orexpression signals directing expression thereof, has been partly orwholly deleted from a chromosomal replicon and/or an extrachromosomalreplicon harboured by said microbial cell.

In a particularly preferred embodiment of the invention, there isprovided a microbial cell wherein the expression of said first and/orsecond expressible enzyme activity is increased, preferablysubstantially increased, whereas the expression of said thirdexpressible enzyme activity is decreased, preferably substantiallydecreased. Reference is made to levels of such increased and decreasedexpression as indicated herein above.

A microbial cell according to the invention, such as a microbial cell,preferably a yeast cell, wherein the expression of said first and/orsecond expressible enzyme activity is increased, preferablysubstantially increased, whereas the expression of said thirdexpressible enzyme activity is decreased, preferably substantiallydecreased, may in one embodiment produce a first metabolite, such ase.g. ethanol, said production of said first metabolite, preferablyethanol, is increased as compared to an expression of said metabolite ina comparable wild-type or isolated cell, said increase is increased by afactor of at least 1.005, for example 1.010, such as 1.015, for example1.020, such as a factor of at least 1.025, for example a factor of atleast 1.030, such as 1.035, for example 1.040, such as 1.045, forexample 1.050, such as 1.055, for example 1.060, such as at least 1.065,for example a factor of at least 1.070, such as 1.075, for example1.080, such as 1.085, for example a factor of at least 1.090, such as1.095, for example 1.100, such as 1.105, for example 1.110, such as1.115, for example 1.120, such as at least 1.125, for example a factorof at least 1.130, such as 1.135, for example 1.140, for example 1.145,such as 1.150, for example 1.155, such as at least 1.160, for example afactor of at least 1.165, such as a factor of at least 1.170, forexample a factor of at least 1.175, such as 1.180, for example 1.185,such as 1.190, for example 1.195, such as a factor of at least 1.200,for example 1.21, such as 1.22, for example 1.23, such as 1.24, forexample 1.25, such as 1.26, for example 1.27, such as 1.28, for example1.29, such as a factor of at least 1.30, for example 1.35, such as 1.40,for example 1.45, such as 1.50, for example 1.55, such as 1.60, forexample 1.65, such as 1.70, for example 1.75, such as 1.80, for example1.85, such as 1.90, for example 1.95, such as a factor of at least 2.0,for example 2.2, such as 2.4, for example 2.6, such as 2.8, for example3.0, such as 3.2, for example 3.4, such as 3.6, for example 3.8, such asat least 4.0, for example 4.2, such as 4.4, for example 4.6, such as4.8, for example at least 5.0, such as 5.2, for example 5.4, such as5.6, for example 5.8, such as at least 6.0, for example 6.2, such as6.4, for example 6.6, such as 6.8, for example at least 7.0, for example7.2, such as 7.4, for example 7.6, such as 7.8, for example 8.0, such as8.2, for example 8.4, such as 8.6, for example 8.8, such as at least9.0, for example 9.2, such as 9.4, for example 9.6, such as 9.8, forexample a factor of at least 10.0.

Said microbial cell, most preferably yeast, having an increasedproduction of a first metabolite has, in another preferred embodiment, adecreased production of a second metabolite, preferably glycerol. Saiddecreased production of said second metabolite, preferably glycerol, isdecreased by at least 0.5 percent, for example at least 1 percent, suchas at least 2 percent, for example 4 percent, such as 6 percent, such asat least 8 percent, for example at least 10 percent, such as 12 percent,for example 14 percent, such as 16 percent, such as at least 18 percent,for example at least 20 percent, such as 22 percent, for example 24percent, such as 26 percent, such as at least 28 percent, for example atleast 30 percent, such as 32 percent, for example 34 percent, such as 36percent, for example 38 percent, such as at least 40 percent, forexample 42 percent, such as 44 percent, for example 46 percent, such as48 percent, such as at least 50 percent, for example 52 percent, such as54 percent, for example 56 percent, such as 58 percent, such as at least60 percent, for example 62 percent, such as 64 percent, for example 66percent, such as 68 percent, such as at least 70 percent, for example 72percent, such as 74 percent, for example 76 percent, such as 78 percent,such as at least 80 percent, for example 82 percent, such as 82 percent,for example 84 percent, such as 86 percent, such as at least 88 percent,for example 90 percent, such as 92 percent, for example 94 percent, suchas 96 percent, for example at least 98 percent, such as 99 percent, forexample 99.2 percent, such as at least 99.4 percent, for example 99.6percent, such as 99.8 percent, for example 99.9 percent, such as 99.92percent, for example 99.94 percent, such as 99.96 percent, for example99.98 percent., such as 99.99 percent, for example an expression levelbeing decreased to such an extend that said expression of said thirdactivity is unassayable using standard state of the art assays and/orsaid expression is effectively repressed and/or substantiallyeliminated.

In another particularly preferred embodiment, the maximum specificgrowth rate of said cell according to the invention is substantiallyunaltered as compared to a comparable wild-type microbial cell or to acomparable isolated microbial cell. However, a microbial cellcharacterised by a decrease in the maximum specific growth rate is alsopreferred according to the invention, such as a microbial cell,preferably a yeast cell, having a maximum specific growth rate that isdecreased by less than 1 percent, such as 1.5 percent, for example 2.0percent, such as by less than 2.5 percent, for example 3.0 percent, suchas 3.5 percent, for example by less than 4.0 percent, such as 4.5percent, for example 5.0 percent, such as by less than 5.5 percent, forexample 6.0 percent, such as 6.5 percent, for example by less than 7.0percent, such as 7.5 percent, for example 8.0 percent, such as by lessthan 8.5 percent, for example 9.0 percent, such as 9.5 percent, forexample by less than 10.0 percent, such as 12 percent, for example 14percent, such as by less than 16 percent, for example 18 percent, suchas 20 percent, for example by less than 25 percent.

In a further embodiment, the microbial cell comprising said first and/orsecond expressible enzyme activity and optionally said third expressibleenzyme activity, if said activity has not been eliminated from said cellby removal, deletion or otherwise, may further optionally comprise afourth expressible enzyme activity. Accordingly, there is provided, inone embodiment of the invention, a microbial cell according to theinvention further comprising said fourth expressible enzyme activitywhereas, in another embodiment, said fourth expressible activity is notpresent in said microbial cell.

Said fourth expressible enzyme activity, when expressed, is controllingan intracellular redox system of said cell, said expression of saidfourth enzyme activity in said microbial cell being either novel oraltered as compared to the expression of said fourth enzyme activity ina comparable wild-type microbial cell or a comparable isolated microbialcell, said fourth expressible enzyme activity being non-identical toeach and all of said first, second and third expressible enzymeactivities.

In a particularly preferred embodiment of the invention, said fourthexpressible enzyme activity is encoded by a nucleotide sequencedesignated SEQ ID NO:1 as illustrated herein below. Said nucleotidesequence was cloned into a multi copy plasmid, Yep24-pPGK. This plasmidwas constructed from Yep24 and contains the promoter and terminator ofPGK and was provided by Mikael Anderlund (Walfridsson et al., 1997). CTHwas ligated into YEp24 behind the strong constitutive promoter of PGKresulting in plasmid Yep24-pPGK-CTH. This plasmid was transferred intostrain TN2 resulting in strain TN4.

The terms altered expression and comparable microbial cell as introducedherein above do also apply to said fourth expressible enzyme activity.The term intracellular redox system shall be understood to comprise anyredox system comprising a coenzyme that is present in correspondingoxidised and reduced forms. Preferred intracellular redox systems arecoenzymes in corresponding oxidised/reduced forms such as e.g. NAD⁺/NADHand NADP⁺/NADPH.

The term maintenance of an intracellular redox system shall beunderstood to comprise the action exerted by any expressible enzymaticactivity which, when expressed, is providing an input to such a systemby e.g. acting in a pathway leading to the synthesis of one or morecomponents of said system or by acting in a recycling or indeed anycyclical reaction involving such components, preferably a reactioninvolving an oxidisation of a reduced coenzyme and/or a reduction of anoxidised coenzyme.

By exerting any one of the above-mentioned actions said fourthexpressible enzyme activity is controlling a redox system. Theabove-described maintenance of said redox system may well lead to anincreased rate of synthesis of any one or more components of saidsystem. Said maintenance may also lead to an increase in the pool of anyone component being comprised in said redox system, notably an increasein the pool of the reduced and/or oxidised form of a coenzyme.

Maintenance shall also be understood to comprise any effect that leadsto a decrease in the pool of components of a redox system, notably adecrease in the pool of the reduced and/or oxidised form of a coenzyme.

The person skilled in the art will know how to assess an increase ordecrease of any form of a coenzyme or of any redox system and he willknow that he must compare the levels of that same coenzyme or redoxsystem in a comparable wild-type microbial cell or an isolated microbialcell grown under identical or substantially similar conditions thatallow for a direct comparison of said levels by exploiting state of theart monitoring techniques such as those described by Weuster andde-Graff (1996) in Adv. Biochem. Eng. Biotechnol., vol. 54, pages 75–108and by Wiechert and de-Graff (1996) in Adv. Biochem. Eng. Biotechnol.,vol. 54, pages 109–154. The person skilled in the art will preferablyanalyse similar or near identical microbial cells with and without saidfourth expressible enzyme activity.

Accordingly, the terms increase and decrease relate to a level ofexpression or synthesis or to a concentration of a coenzyme and/or aredox system. The term level is used interchangeably in the art withterms such a synthesis rate and concentration. The person skilled in theart will be familiar with such terms and attach the correct meaning totheir use in different contexts.

By acting to increase and/or decrease the rate of synthesis and/or thepool of a redox system component, the fourth expressible enzyme activityis controlling said intracellular redox system. The term alteration inthis context shall be understood to comprise any change or deviationfrom the presence and/or amount of a redox system present in acomparable wild-type microorganism or an a comparable isolated microbialcell.

Any redox system can generally be perceived to contribute to theprovision of a certain redox level in a cell. The totality of all suchredox systems in a cell determines the redox level of said cell. Theredox level of a cell thus comprises the presence and/or amount of thetotality of a reducing power and an oxidising power present in saidcell. Accordingly, an alteration of an intracellular redox system can bemeasured either by monitoring the increase or decrease of a specificredox system, i.e. an increase or decrease in both the oxidised form aswell as in the reduced form of a coenzyme constituting said redoxsystem, or alternatively, said alteration can be monitored by measuringan overall cellular redox level.

In one preferred embodiment according to the invention, said fourthexpressible enzyme activity provides the means for transfering reducingor oxidizing potential from one redox system to another. Alteration ofan intracellular redox system may thus result in an increase or decreasein either the oxidised form or the reduced form of a coenzyme. It mayalso result in either one of those forms being increased while the otherform is decreased. Any such alteration can be measured either bymonitoring the increase or decrease of any one specific redox system,i.e. an increase or decrease in either an oxidised form and/or a reducedform of said coenzyme. Alternatively, said alteration can be monitoredby measuring an overall intracellular redox level.

The person skilled in the art will be aware of such alterations leadingto an increased or decreased redox level and, as exemplified herein andexplained further in detail below, the skilled person will be aware ofthe state of the art techniques available for monitoring anintracellular redox level.

In one embodiment therefore, when said fourth expressible enzymeactivity is present in said microbial cell and expressed therein, itresults in an increased or decreased level, preferably an increasedlevel, of at least one intracellular coenzyme in its oxidised or reducedform. Said coenzyme in its oxidised/reduced form is preferably selectedfrom the group consisting of FAD/FADH₂, NAD⁺/NADH and NADP⁺/NADPH.

Accordingly, the level of at least one intracellular coenzyme in itsoxidised or reduced form is either increased or decreased, preferablyincreased, by a factor of at least 1.005, for example 1.010, such as1.015, for example 1.020, such as a factor of at least 1.025, forexample a factor of at least 1.030, such as 1.035, for example 1.040,such as 1.045, for example 1.050, such as 1.055, for example 1.060, suchas at least 1.065, for example a factor of at least 1.070, such as1.075, for example 1.080, such as 1.085, for example a factor of atleast 10.090, such as 1.095, for example 1.100, such as 1.105, forexample 10.110, such as 1.115, for example 1.120, such as at least1.125, for example a factor of at least 1.130, such as 1.135, forexample 1.140, for example 1.145, such as 1.150, for example 1.155, suchas at least 1.160, for example a factor of at least 1.165, such as afactor of at least 1.170, for example a factor of at least 1.175, suchas 1.180, for example 1.185, such as 1.190, for example 1.195, such as afactor of at least 1.200, for example 1.21, such as 1.22, for example1.23, such as 1.24, for example 1.25, such as 1.26, for example 1.27,such as 1.28, for example 1.29, such as a factor of at least 1.30, forexample 1.35, such as 1.40, for example 1.45, such as 1.50, for example1.55, such as 1.60, for example 1.65, such as 1.70, for example 1.75,such as 1.80, for example 1.85, such as 1.90, for example 1.95, such asa factor of at least 2.0, for example 2.2, such as 2.4, for example 2.6,such as 2.8, for example 3.0, such as 3.2° for example 3.4, such as 3.6,for example 3.8, such as at least 4.0, for example 4.2, such as 4.4, forexample 4.6, such as 4.8, for example at least 5.0, such as 5.2, forexample 5.4, such as 5.6, for example 5.8, such as at least 6.0, forexample 6.2, such as 6.4, for example 6.6, such as 6.8, for example atleast 7.0, for example 7.2, such as 7.4, for example 7.6, such as 7.8,for example 8.0, such as 8.2, for example 8.4, such as 8.6, for example8.8, such as at least 9.0, for example 9.2, such as 9.4, for example9.6, such as 9.8, for example a factor of at least 10.0.

Although an increase is preferred, it shall be understood that the termalteration is by no means limited to an increase in the level of atleast one intracellular coenzyme in its oxidised or reduced form. Saidalteration shall also comprise any decrease in the level of at least oneintracellular coenzyme in its oxidised or reduced form.

In an embodiment of the invention wherein the expression of said fourthexpressible enzyme activity results in an increase or a decrease in thelevel i.e. concentration of an intracellular redox system as a whole,i.e. an increase or decrease of both of two corresponding oxidised andreduced forms of a coenzyme, said alteration is an increase or decrease,preferably an increase, by a factor of at least 1.005, for example1.010, such as 1.015, for example 1.020, such as a factor of at least1.025, for example a factor of at least 1.030, such as 1.035, forexample 1.040, such as 1.045, for example 1.050, such as 1.055, forexample 1.060, such as at least 1.065, for example a factor of at least1.070, such as 1.075, for example 1.080, such as 1.085, for example afactor of at least 1.090, such as 1.095, for example 1.100, such as1.105, for example 1.110, such as 1.115, for example 1.120, such as atleast 1.125, for example a factor of at least 1.130, such as 1.135, forexample 1.140, for example 1.145, such as 1.150, for example 1.155, suchas at least 1.160, for example a factor of at least 1.165, such as afactor of at least 1.170, for example a factor of at least 1.175, suchas 1.180, for example 1.185, such as 1.190, for example 1.195, such as afactor of at least 1.200, for example 1.21, such as 1.22, for example1.23, such as 1.24, for example 1.25, such as 1.26, for example 1.27,such as 1.28, for example 1.29, such as a factor of at least 1.30, forexample 1.35, such as 1.40, for example 1.45, such as 1.50, for example1.55, such as 1.60, for example 1.65, such as 1.70, for example 1.75,such as 1.80, for example 1.85, such as 1.90, for example 1.95, such asa factor of at least 2.0, for example 2.2, such as 2.4, for example 2.6,such as 2.8, for example 3.0, such as 3.2, for example 3.4, such as 3.6,for example 3.8, such as at least 4.0, for example 4.2, such as 4.4, forexample 4.6, such as 4.8, for example at least 5.0, such as 5.2, forexample 5.4, such as 5.6, for example 5.8, such as at least 6.0, forexample 6.2, such as 6.4, for example 6.6, such as 6.8, for example atleast 7.0, for example 7.2, such as 7.4, for example 7.6, such as 7.8,for example 8.0, such as 8.2, for example 8.4, such as 8.6, for example8.8, such as at least 9.0, for example 9.2, such as 9.4, for example9.6, such as 9.8, for example a factor of at least 10.0.

Said fourth expressible enzyme activity may well result in an increaseor a decrease, preferably an increase, of more than one intracellularredox system. It shall be understood that in one embodiment of theinvention, said fourth expressible enzyme activity, when expressed, isresulting in an increased level of at least one intracellular redoxsystem.

In a particularly preferred aspect of the invention, said fourthexpressible enzyme activity is an intracellular transhydrogenaseactivity, preferably a pyridine nucleotide transhydrogenase activity,and more preferably a pyridine nucleotide transhydrogenase activity suchas that of CTH of Azotobacter vinelandii as harboured by Saccharomycescerevisiae TN4 deposited under DSM Accession Number 12267, or afunctionally equivalent activity. The term functional equivalentactivity is defined herein above and does also apply to the context inwhich the term is used here.

The pyridine nucleotide transhydrogenase activity is either endogenousor heterologous to said microbial cell wherein it is expressed and saidactivity is either exhibited by a polypeptide which is membrane-bound ina natural host organism or located i.e. present in the cytoplasm of anatural host organism, said natural host organism preferably beingselected from the group of mammalian cells, plant cells, eukaryotic andprokaryotic cells including microbial and bacterial cells such as e.g.Gram-positive microbial prokaryote and a Gram-negative microbialprokaryote.

Expression of said pyridine nucleotide transhydrogenase activity in saidmicrobial cell in one preferred embodiment of the invention results inan increased conversion of NADPH and NAD to NADH and NADP. In anotherembodiment said expression of said pyridine nucleotide transhydrogenaseactivity results in an increased consumption of NADPH and an increasedformation of NADH. In another embodiment the expression has the effectof increasing the consumption of NADH and increasing the formation ofNADPH. In vet another embodiment the expression results in an increasedformation of NADH and/or NADP. However, said expression may also resultin a decreased formation of NADPH and/or a decreased NADPH/NADP retio.The pyridine nucleotide transhydrogenase activity is preferablyexpressed in said microbial cell under anaerobic growth conditions andpreferably results in the ratio of NADPH/NADP being lower than the ratioof NADH/NAD.

In another preferred embodiment, the transhydrogenase activity,preferably an activity that is membrane-bound in a natural hostorganism, is inserted into the plasma membrane of a microbial cell,preferably a yeast cell. The transhydrogenase activity would mediate areaction consuming NADP and generating NADPH. In a particularlypreferred embodiment, an E. coli transhydrogenase is expressed in ayeast cell and leads to an increased level of NADPH. The expression ofthe E. coli transhydrogenase is coupled to a proton gradient across themembrane of the natural host organism and a similar coupling is likelyto be established when the membrane-bound E. coli transhydrogenaseenzyme is integrated into the plasma membrane of a yeast cell such ase.g. Saccharomyces cerevisiae.

In a particularly preferred embodiment, the fourth expressible enzymeactivity is that of CTH comprised in Saccharomyces cerevisiae strain TN4deposited under Accession Number DSM 12267.

The microbial cell according to invention is preferably one suitable forstorage in the form of a frozen or freeze-dried preparation such as alyophilisate from which the microbial cell is partly or whollyreconstitutable.

In yet another embodiment of the invention, the microbial cell has beenmetabolically engineered as described above and is capable ofalternative NADH re-oxidation. Said alternative NADH re-oxidation ismediated at least by the combined expression of said above-mentionedfirst and second expressible enzyme activities. In one preferredembodiment said alternative NADH-reoxidation is mediated byoverexpression of said first and second expressible enzyme activities ina microbial cell having a substantially decreased expression of saidthird expressible enzyme activity, or a microbial cell wherein saidexpression has been repressed or eliminated or deleted. Said fourthexpressible enzyme activity may optionally be expressed concomitantlywith an overexpression of said first and second expressible enzymeactivities and a substantially reduced and preferably eliminatedexpression of said third expressible enzyme activity. In one preferredembodiment of the invention, said first, second, third and fourthexpressible enzyme activities are those of a glutamate synthase, aglutamine synthetase, a glutamate dehydrogenase and a transhydrogenase,respectively.

Alternative NADH re-oxidation shall be understood to comprise theintroduction of a novel major pathway for NADH re-oxidation or ageneration of a substantially altered pathway for NADH-reoxidation in amicrobial cell. Alteration in respect of a pathway for alternative NADHoxidation shall be understood in the context of the rate of a reactionmediating a conversion of one metabolite to another, said reaction alsoresulting in NADH re-oxidation. The rate of said re-oxidation reactionin a microbial cell capable of alternative NADH re-oxidation issubstantially increased as compared to the rate of said reaction in acomparable microbial cell. The definition of the term comparable inrespect of microbial cells is already introduced herein above.

Alternative NADH re-oxidation is an example of a microbial cell whereinthe expression of a transhydrogenase, in combination with severaladditionally preferred expressible enzyme activities, are capable ofgenerating a purposeful redesigning of a complex network of metabolicreactions. The redesigned microbial cell is invented by replacing orsupplementing a normally dominant first metabolic reaction with a secondreaction that is normally insignificant in relation to reaction rateand/or product formation as compared to said first dominant reaction.However, by significantly increasing said second reaction while at thesame time significantly decreasing or even eliminating said firstreaction, it is possible to achieve an alternative NADH re-oxidation. Ina further embodiment of the invention, there is also provided amicrobial cell capable of alternative NADPH re-oxidation or alternativeNADP reduction.

In another aspect of the invention, there is provided a compositioncomprising the microbial cell and a carrier, preferably aphysiologically acceptable carrier and more preferably a water-basedliquid such as a broth suitable for culturing said microbial cell. Thecomposition in a preferred embodiment is a fermentation starter culture.

There is also provided the aspect of a nucleotide sequence encoding anovel and industrially relevant transhydrogenase enzyme activity, saidsequence comprising SEQ ID NO:1, as illustrated herein below, or partthereof, including functionally equivalent derivatives thereof encodinga transhydrogenase enzyme activity, preferably but not limited toconservative nucleotide substitutions and/or nucleotide deletions and/ornucleotide insertions. Said functionally equivalent derivatives may thusbe at least 99.9 percent identical to SEQ ID NO:1, such as at least 99.8percent identical to SEQ ID NO:1, for example at least 99.7 percentidentical, such as at least 99.6 percent identical, for example at least99.5 percent identical, such as at least 99.4 percent identical, forexample at least 99.3 percent identical, such as at least 99.2 percentidentical, for example at least 99.1 percent identical, such as at least99 percent identical to SEQ ID NO:1, for example at least 98.5 percentidentical to SEQ ID NO:1, such as at least 98.0 percent identical, forexample 97.5 percent identical, such as at least 97.0 percent identicalto SEQ ID NO:1, for example at least 96.5 percent identical, such as atleast 96.0 percent identical, for example at least 95.5 percentidentical, such as at least 95.0 percent identical, for example at least94.5 percent identical, such as at least 94.0 percent identical, forexample at least 93.5 percent identical, such as at least 93.0 percentidentical to SEQ ID NO:1, for example at least 92.5 percent identical,such as at least 92.0 percent identical, for example at least 91.5percent identical, such as at least 91.0 percent identical, for exampleat least 90.5 percent identical, such as at least 90.0 percentidentical, for example at least 85.0 percent identical to SEQ ID NO:1.In one embodiment, SEQ ID NO:1 is a sequence that is synthesised partlyor wholly in vitro.

In a further aspect of the invention there is provided a recombinantDNA-replicon in the form of a vector comprising the nucleotide sequencedesignated SEQ ID. NO: 1 including functionally equivalent derivatives.The nucleotide sequence designated SEQ ID. NO: 1 is preferably operablylinked to an expression signal comprised in said replicon, saidexpression signal directing expression of said nucleotide sequence.There is also provided a microbial cell microbial cell harbouring thenucleotide sequence designated SEQ ID NO:1 or a recombinant replicon inthe form of a vector harbouring said nucleotide sequence.

The recombinant DNA-replicon is preferably one capable of replicating ina yeast cell and/or in a prokaryotic microbial cell such as a lacticacid bacterial cell. Preferred yeast vectors comprise a selectablemarker and one or more sites in a nucleotide sequence specific for arestriction endonuclease. An autonomously replicating sequence (ARS)mediates replication of said replicon when harboured in a yeast cell.The vector is preferably based on a plasmid selected from the groupconsisting of a yeast episomal plasmid (Yep), such as the 2 μm plasmid,a yeast replicating plasmid (Yrp), a yeast centromeric plasmid (Ycp) anda yeast-integrating plasmid (Yip). A particularly preferred replicon isthe one harboured by Saccharomyces cerevisiae strain TN4 deposited underAccession Number DSM 12267.

Vectors capable of being maintained in a prokaryotic microbial cell suchas a lactic acid bacterial cell are well described in the literature andpreferably contain a replicon directing e.g. rolling circle replicationor θ-replication, a selectable marker such as a nonsense mutationpreventing selection and/or replication unless suppressed by asuppresser comprised by a cell comprising said vector, and one or moresites cleavable by a restriction endonuclease.

In a further aspect of the invention, there is provided an amino acidsequence encoded by the nucleotide sequence designated SEQ ID NO: 1, orany functionally equivalent derivative thereof, said amino acid sequencecomprising the sequence SEQ ID NO:2, as illustrated herein below, or apart thereof, including any functionally equivalent derivativesexhibiting transhydrogenase activity, preferably, but not limited to,functionally equivalent derivatives comprising conservative amino acidsubstitutions.

Said functionally equivalent derivative of said amino acid sequencedesignated SEQ ID NO: 2 may thus be at least 99 percent identical to SEQID NO:2, such as at least 98 percent identical to SEQ ID NO:2, forexample at least 97 percent identical, such as at least 96 percentidentical, for example at least 95 percent identical, such as at least94 percent identical, for example at least 93 percent identical, such asat least 92 percent identical, for example at least 91 percentidentical, such as at least 90 percent identical to SEQ ID NO:2, forexample at least 89 percent identical to SEQ ID NO:2, such as at least88 percent identical, for example 87 percent identical, such as at least86 percent identical to SEQ ID NO:2, for example at least 85 percentidentical to SEQ ID NO:2. In one embodiment, SEQ ID NO:2 is a sequencethat is synthesised partly or wholly in vitro.

In a further aspect of the invention there is provided a microbial cell,preferably a yeast cell or a bacterial cell, or a composition comprisingsaid cell, for use in a production of a first metabolite such as aprimary or secondary metabolite, preferably a primary metabolite andmore preferably an alcohol or an acid, such as e.g. ethanol, glycerol,acetic acid and propionic acid, ethanol being particularly preferred.

When the first metabolite is a secondary metabolite, said secondarymetabolite is preferably selected from the group of secondarymetabolites consisting of a β-lactam, a polyketide, a terpene, asteroid, a quinone, a coumarin, a flavonoid, an alkaloid, a piperidine,a pyridine, and the like.

Said production of said first metabolite is preferably substantiallyincreased as compared to the production of said first metabolite in acomparable wild-type cell or a comparable isolated microbial cell.Accordingly, said microbial cell production of said first metabolite isincreased at least by a factor of 1.02, such as a factor of at least1.04, for example 1.06, such as 1.08, for example 1.10, such as at least1.12, for example 1.14, such as 1.16, for example 1.18, such as 1.2, forexample 1.25, such as 1.3, for example 1.4, such as 1.5, for example1.6, such as 1.7, for example 1.8, such as 1.9, for example 2.0, such as2.25, for example 2.5, such as 3, for example 3.5, such as a factor ofat least 4, for example 4.5, such as 5, for example 6, such as 7, forexample 8, such as 9, for example 10, such as 15, for example 20, suchas 25, for example 30, such as 35, for example 40, such as 50, forexample 60, such as 80, for example at least 100, such as 150, forexample 200, such as 250, for example 300, such as 350, for example 400,such as 500, for example 600, such as 800, for example at least 1000,such as 1500, for example 2000, such as 2500, for example 3000, such as3500, for example 4000, such as at least 5000, for example 6000, such as8000, for example at least 10000, such as 15000, for example 20000, suchas at least 25000, for example 30000, such as 35000, for example 40000,such as a factor of at least 50000.

It may furthermore be advantageous to produce and/or purify by means ofany state of the art down-stream processing technique said firstmetabolite in an organism such as e.g. a fungal cell, a yeast cell or abacterial cell. Any of said eukaryotic or prokaryotic cells for use insaid production preferably qualify for GRAS status (“Generally RegardedAs Safe”) with the Federal Drug Administration of the United States ofAmerica.

In another embodiment, there is provided a microbial cell, preferably ayeast cell, for use in said production of said first metabolite, saidcell further producing a second metabolite, preferably glycerol, saidproduction of said second metabolite being substantially decreased ascompared to the production of said second metabolite in a comparablewild-type cell or a comparable isolated microbial cell.

Accordingly, said production of said second metabolite is decreased byat least 2 percent, such as 4 percent, for example at least 6 percent,such as 8 percent, for example at least 10 percent, such as 12 percent,for example 14 percent, such as 16 percent, for example 18 percent, suchas at least 20 percent, for example 24 percent, such as at least 30percent, for example 35 percent, such as at least 40 percent, forexample 50 percent, such as 60 percent, for example at least 70 percent,such as 80 percent, for example at least 90 percent, such as decreasedby at least 92 percent, for example 94 percent, such as 96 percent, forexample 98 percent, such as decreased by 99 percent or decreased to suchan extent that said second metabolite is virtually unassayable usingstate of the art assays for identifying and/or quantifying said secondmetabolite.

When the microbial cell is a prokaryotic cell such as e.g. a lactic acidbacterial cell for use in the production of a first metabolite, saidfirst metabolite is selected from the group consisting of lactic acidand an aroma component such as acetoin, acetaldehyde, 2,3-butyleneglycol, or diacetyl.

In one embodiment, the microbial cell or the composition according tothe invention is preferably used in a production of a first metaboliteor used in a method of generating alternative intracellular NADHre-oxidation. Accordingly, the microbial cell is providing a novel or,in terms of efficiency and/or overall rate of reaction, a much improvedpathway for alternative NADH re-oxidation for the purpose of providing,supplementing and/or increasing a pool of intracellular NAD, saidprovision, supplementation and/or increase being used in a process ofaltering, directing and/or redirecting the flux of primary and/orsecondary metabolites in said cell.

In another embodiment there is provided a microbial cell for use in aproduction of a first metabolite, said cell harbouring a novel or, interms of efficiency and/or overall rate of reaction, a much improvedpathway for alternative NAD reduction for the purpose of providing,supplementing and/or increasing a pool of intracellular NADH, saidprovision, supplementation and/or increase being used in a process ofaltering, directing and/or redirecting the flux of primary and/orsecondary metabolites in said cell.

In yet another embodiment of the invention, there is provided amicrobial cell for use in the production of a first metabolite, saidcell harbouring a novel or, in terms of efficiency and/or overall rateof reaction, a much improved pathway for alternative NADPH re-oxidationfor the purpose of providing, supplementing and/or increasing a pool ofintracellular NADP, said provision, supplementation and/or increasebeing used in a process of altering, directing and/or redirecting theflux of primary and/or secondary metabolites in said cell.

In a still further embodiment of the invention, there is provided amicrobial cell for use in the production of a first metabolite, saidcell harbouring a novel or, in terms of efficiency and/or overall rateof reaction, a much improved pathway for alternative NADP reduction forthe purpose of providing, supplementing and/or increasing a pool ofintracellular NADPH, said provision, supplementation and/or increasebeing used in a process of altering, directing and/or redirecting theflux of primary and/or secondary metabolites in said cell.

In a yet further aspect of the invention there is provided a microbialcell according to invention for use in a preparation of a drinkable oran edible product. There is also provided a microbial cell for use in aproduction of a first metabolite for use in a drinkable or an edibleproduct, preferably a product having desirable organoleptic qualities.In one embodiment, said first metabolite has and/or provides a desirableorganoleptic quality to said product. In a particularly preferredembodiment, said first metabolite is ethanol.

The microbial cell for use in a production of a first metaboliteaccording to the invention, in one embodiment, further produces a secondmetabolite, the production of said second metabolite being substantiallydecreased as compared to the production of said second metabolite in acomparable wild-type cell or a comparable isolated microbial cell, saiddecreased production resulting in a provision of a desirableorganoleptic quality to said product. In a further embodiment saidproduct is a functional food.

In yet another aspect of the invention there is provided the use of amicrobial cell or a composition in a production of a first metabolite,said metabolite being a primary metabolite or a secondary metabolite, ametabolite endogenous to said microbial cell or a gene productheterologous to said microbial cell.

In a preferred embodiment there is provided a use of a microbial cellwherein said production of said first metabolite is substantiallyincreased as compared to the production of said first metabolite in acomparable wild-type cell or a comparable isolated microbial cell. Saidproduction of said first metabolite is increased at least by a factor of1.01, such as 1.02, for example 1.03, such as a factor of at least 1.04,for example 1.05, such as 1.06, for example 1.07, such as 1.08, forexample 1.09, such as 1.10, for example 1.11, such as at least 1.12, forexample 1.14, such as 1.16, for example 1.18, such as 1.2, for example1.25, such as 1.3, for example 1.4, such as 1.5, for example 1.6, suchas 1.7, for example 1.8, such as 1.9, for example 2.0, such as 2.25, forexample 2.5, such as 3, for example 3.5, such as a factor of at least 4,for example 4.5, such as 5, for example 6, such as 7, for example 8,such as 9, for example 10, such as 15, for example 20, such as 25, forexample 30, such as 35, for example 40, such as 50, for example 60, suchas 80, for example at least 100, such as 150, for example 200, such as250, for example 300, such as 350, for example 400, such as 500, forexample 600, such as 800, for example at least 1000, such as 1500, forexample 2000, such as 2500, for example 3000, such as 3500, for example4000, such as at least 5000, for example 6000, such as 8000, for exampleat least 10000, such as 15000, for example 20000, such as at least25000, for example 30000, such as 35000, for example 40000, such as afactor of at least 50000.

It is preferred that the microbial cell is a yeast cell or a prokaryoticmicrobial cell and that said first metabolite is an alcohol or an acid,preferably ethanol, acetic acid, lactic acid or propionic acid.

In a preferred use there is provided a microbial cell, preferably ayeast cell, further producing a second metabolite, said production ofsaid second metabolite being substantially decreased as compared to theproduction of said second metabolite in a comparable wild-type cell or acomparable isolated microbial cell. Particularly preferred is a usewherein said second metabolite is glycerol or an undesirable aromacomponent naturally produced by a lactic acid bacterial cell.

The production of said second metabolite, preferably glycerol or anundesirable aroma component produced by a lactic acid bacterial cell, isreduced in a preferred use of said microbial cell by at least by atleast 2 percent, such as 4 percent, for example at least 6 percent, suchas 8 percent, for example at least 10 percent, such as 12 percent, forexample 14 percent, such as 16 percent, for example 18 percent, such asat least 20 percent, for example 24 percent, such as at least 30percent, for example 35 percent, such as at least 40 percent, forexample 50 percent, such as 60 percent, for example at least 70 percent,such as 80 percent, for example at least 90 percent, such as decreasedby at least 92 percent, for example 94 percent, such as 96 percent, forexample 98 percent, such as decreased by 99 percent or decreased to suchan extent that said second metabolite is virtually unassayable usingstate of the art assays for identifying and/or quantifying said secondmetabolite.

Another preferred use of said microbial cell is in preparation of adrinkable or edible product or in a production of a first metabolite foruse in said drinkable or edible product, said first metabolite havingand/or providing a desirable organoleptic quality to said product.Preferably the first metabolite is ethanol or, when the microbial cellis a lactic acid bacterial cell, an aroma component produced by saidlactic acid bacterial cell, preferably acetoin and/or diacetylactis.

A much preferred use of said microbial cell in said preparation of saiddrinkable or edible product is that of a microbial cell according to theinvention, preferably a yeast cell or a lactic acid bacterial cell,further producing a second metabolite, said production of said secondmetabolite being substantially decreased as compared to the productionof said second metabolite in a comparable wild-type cell or a comparableisolated microbial cell, said decreased production resulting in aprovision of a desirable organoleptic quality to said product, saiddecrease is at least 2 percent, such as 4 percent, for example at least6 percent, such as 8 percent, for example at least 10 percent, such as12 percent, for example 14 percent, such as 16 percent, for example 18percent, such as at least 20 percent, for example 24 percent, such as atleast 30 percent, for example 35 percent, such as at least 40 percent,for example 50 percent, such as 60 percent, for example at least 70percent, such as 80 percent, for example at least 90 percent, such asdecreased by at least 92 percent, for example 94 percent, such as 96percent, for example 98 percent, such as decreased by 99 percent ordecreased to such an extent that said second metabolite is virtuallyunassayable using state of the art assays for identifying and/orquantifying said second metabolite.

There is also provided a use of a microbial cell, preferably a yeastcell or a lactic acid bacterial cell, in a preparation of a functionalfood.

In a yet further aspect of the invention there is provided a method ofproducing a first metabolite, said method comprising the steps of

-   -   i) cultivating a microbial cell in a suitable growth medium and        under such conditions that said microbial cell is producing a        first metabolite and optionally    -   ii) isolating said first metabolite in a suitable form, and        further optionally    -   iii) purifying said isolated first metabolite.

The method comprises the culturing of any microbial cell including amicrobial eukaryote and a microbial prokaryote. Among microbialeukaryotes, yeast cells and fungal cells are preferred, such as yeastcells like e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris and the like, as well as algae such as e.g.Chlamydomonas reinhardi, slime moulds such as e.g. Dictyosteliumdiscoideum and filamentous fungi. Preferred filamentous fungi accordingto the method are species of Neurospora and Aspergillus such as e.g.Neurospora crassa, Aspergillus nidulans, Aspergillus niger, Aspergilluscryzae and Penicillium chrysogenum. Particularly preferred are manyindustrially relevant yeast cells, slime moulds and filamentous fungiproviding a source of production of products such as e.g. antibiotics,steroids, pigments, enzymes, organic alcohols and acids, amino acids,polysaccharides and the like.

The method also pertains to the culturing of microbial prokaryotes suchas Gram-positive species such as e.g. Bacillus subtilis, Bacillusthuringensis, Bacillus licheniformis, Bacillus lentus and Bacilusstearothermophilus and Gram-negative species such as Escherichia coli.Particularly preferred are also lactic acid bacterial species such ase.g. Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactococcuslactis subsp. cremoris, Lactococcus lactis subsp. diacetylactis,Leuconostoc species, Lactobacillus species, Pediococcus species andsimilar industrially relevant species like e.g. Bifidobacterium.

Embodiments of this aspect of the invention comprise a method whereinsaid first metabolite is either a primary metabolite or a secondarymetabolite. The metabolite may be produced in a cell also capable offurther producing e.g. an endogenous or a heterologous product selectedfrom the group consisting a protease, an amylase, a cellulase, aβ-glucanase, an endoglucanase, a phosphatase, a xylanase, a lipase, aβ-lactamase, a β-galactosidase, a βglucoronidase, and a xylosidase. Whenthe microbial cell is a lactic acid bacterium, said metabolite ispreferably diacetyl, acetoin, or lactic acid.

The method according to the invention pertains in one embodiment to anincreased production of said first metabolite, such as a substantiallyincreased production, as compared to the production of said firstmetabolite in a comparable wild-type cell or a comparable isolatedmicrobial cell. Accordingly, there is provided a method by which saidproduction of said first metabolite is increased at least by a factor of1.01, such as 1.02, for example 1.03, such as a factor of at least 1.04,for example 1.05, such as 1.06, for example 1.07, such as 1.08, forexample 1.09, such as 1.10, for example 1.11, such as at least 1.12, forexample 1.14, such as 1.16, for example 1.18, such as 1.2, for example1.25, such as 1.3, for example 1.4, such as 1.5, for example 1.6, suchas 1.7, for example 1.8, such as 1.9, for example 2.0, such as 2.25, forexample 2.5, such as 3, for example 3.5, such as a factor of at least 4,for example 4.5, such as 5, for example 6, such as 7, for example 8,such as 9, for example 10, such as 15, for example 20, such as 25, forexample 30, such as 35, for example 40, such as 50, for example 60, suchas 80, for example at least 100, such as 150, for example 200, such as250, for example 300, such as 350, for example 400, such as 500, forexample 600, such as 800, for example at least 1000, such as 1500, forexample 2000, such as 2500, for example 3000, such as 3500, for example4000, such as at least 5000, for example 6000, such as 8000, for exampleat least 10000, such as 15000, for example 20000, such as at least25000, for example 30000, such as 35000, for example 40000, such as afactor of at least 50000.

When being isolated or when being isolated and purified, said metaboliteis isolated or isolated and purified according to any available state ofthe art techniques for isolating or isolating and purifying ametabolite.

In one preferred embodiment of the invention, the method pertains to theproduction in a yeast cell or in a lactic acid bacterial cell of a firstmetabolite such as a primary or secondary metabolite, preferably aprimary metabolite and more preferably an alcohol or an acid, such ase.g. ethanol, glycerol, acetic acid and propionic acid, with ethanolbeing particularly preferred. In a much preferred embodiment, themicrobial cell according to the method is a yeast cell and the firstmetabolite is ethanol. When said first metabolite is a secondarymetabolite, said secondary metabolite is preferably selected from thegroup of secondary metabolites consisting of a β-lactam, a polyketide, aterpene, a steroid, a quinone, a coumarin, a flavonoid, an alkaloid, apiperidine, a pyridine, and the like.

In another embodiment of the method according to the invention, there isprovided a microbial cell, preferably a yeast cell or a lactic acidbacterial cell, said cell further producing a second metabolite, theproduction of said second metabolite being substantially decreased ascompared to the production of said second metabolite in a comparablewild-type cell or a comparable isolated microbial cell. In oneembodiment, said decrease of said production of said second metaboliteis at least 2 percent, such as 4 percent, for example at least 6percent, such as 8 percent, for example at least 10 percent, such as 12percent, for example 14 percent, such as 16 percent, for example 18percent, such as at least 20 percent, for example 24 percent, such as atleast 30 percent, for example 35 percent, such as at least 40 percent,for example 50 percent, such as 60 percent, for example at least 70percent, such as 80 percent, for example at least 90 percent, such asdecreased by at least 92 percent, for example 94 percent, such as 96percent, for example 98 percent, such as decreased by 99 percent ordecreased to such an extent that said second metabolite is virtuallyunassayable using state of the art assays for identifying and/orquantifying said second metabolite.

In one preferred embodiment the second metabolite is glycerol when saidcell is a yeast cell having a substantially increased production of afirst metabolite, preferably ethanol. In another preferred embodiment,when the cell is a lactic acid bacterial cell, the second metabolite isan undesirable aroma component naturally produced by a lactic acidbacterial cell.

In one embodiment of the method according to the invention the microbialcell is a lactic acid bacterial cell and said first metabolite isselected from the group consisting of lactic acid and a desirable aromacomponent such as acetoin and diacetyl. In another embodiment, themicrobial cell is a Gram-positive bacterial cell, preferably a cellcapable of producing an enzyme such as e.g. a protease, an amylase, acellulase, a β-glucanase, an endoglucanase, a phosphatase, a xylanase, alipase, a β-lactamase, a β-galactosidase or a xylosidase.

There is also provided a method for generating an alternativere-oxidation of a reduced coenzyme, said method, in one embodiment,consisting essentially of providing in a microbial cell a novel or, interms of efficiency and/or overall rate of reaction, a much improvedpathway for alternative NADH and/or NADPH re-oxidation for use inproviding, supplementing and/or increasing a pool of intracellular NADand/or NADP, said provision, supplementation and/or increase being usedin a process of altering, directing and/or redirecting the flux ofprimary and/or secondary metabolites in said cell.

Also, there is provided a method for generating an alternative reductionof an oxidised coenzyme, said method consisting essentially of providingin a microbial cell a novel or, in terms of efficiency and/or overallrate of reaction, a much improved pathway for alternative NAD and/orNADP reduction for the purpose of providing, supplementing and/orincreasing a pool of intracellular NADH and/or NADPH, said provision,supplementation and/or increase being used in a process of altering,directing and/or redirecting the flux of primary and/or secondarymetabolites in said cell.

In a yet further aspect of the invention, there is provided a method ofconstructing a microbial cell according to the invention, said methodcomprising the steps of

-   -   i) operably linking a nucleotide sequence encoding said first        expressible enzyme activity with an expression signal not        natively associated with said nucleotide sequence, and/or    -   ii) operably linking a nucleotide sequence encoding said second        expressible enzyme activity with an expression signal not        natively associated with said nucleotide sequence, and    -   iii) eliminating said third expressible enzyme activity from        said microbial cell, or optionally operably linking a nucleotide        sequence encoding said third expressible enzyme activity with an        expression signal not natively associated with said nucleotide        sequence, said expression signal generating a reduced expression        of said nucleotide sequence, and    -   iv) introducing said operably linked nucleotide sequences        obtained under i) and iii), and optionally the nucleotide        sequence obtained under ii), into said microbial cell, or    -   v) introducing said operably linked nucleotide sequence obtained        under i), and optionally the nucleotide sequence obtained under        ii), into said microbial cell obtained under iii) wherein said        third expressible enzyme activity has been eliminated.

Said expression signal may direct a substantially constitutiveexpression, a constitutive α-pression during growth of said cell in aparticular growth phase, an inducible expression in response to thepresence and/or level of an inducer or the absence and/or level of arepressor. The expression signal is preferably a regulatable expressionsignal such as a regulatable transcription initiation signal and/or aregulatable translational initiation signal, such as an expressionsignal regulatable in response to an alteration in a value, level and/orconcentration of a factor such as a physiological growth parameter,preferably a parameter selected from the group consisting of pH,temperature, salt content including osmolarity, anaerobicity, aerobicityincluding oxygen level, energy level including a membrane potential anda proton motive force.

The expression signal is preferably a promoter being either growth phaseregulated, inducible and/or repressible and/or, in a natural hostorganism, directing expression of a gene encoding a gene productinvolved in mediating a reaction of a biosynthetic pathway and/or amajor metabolic pathway, preferably a pathway selected from the group ofpathways consisting of glycolysis, gluconeogenesis, citric acid cycle,and pentose phosphate pathway.

The expression signal may be further regulated by an upstream activatingsequence (UAS), by an enhancer element or by a silencer element. Theperson skilled in the art will be aware of general molecular biologytechniques for use in the construction in vitro of a recombinant DNAmolecule. Such techniques are described e.g. in Sambrook et al. (1989)and in Old and Primrose (ibid.). Said skilled artisan will further beaware of the academic literature including general textbooks onmolecular biology and genetic engineering and he will be able to combinevarious expression signals such as putative or recognised promoterregions with a range of regulatory nucleotide sequences generally knownto exert an effect on the level of gene expression. The skilled personis able to monitor gene expression by construction of suitabletranscriptional and/or translational fusions of an expression signal toa reporter gene generally available in the art. An expression signal canbe a cloned expression signal or an in vitro synthesised expressionsignal. Expression signals in prokaryotic microbial cells are known tocomprise so-called -35 and -10 regions and numerous examples of suchregions are available from various databases.

Expression signals may be optimised by increasing the promoter strength,by adjusting translational initiation sequences, by optimising thechoice of codons by using so-called highly expressed codons, byadjusting the secondary structure of the mRNA, by increasing theefficiency of transcriptional termination, by increasing or decreasing acopy number of a vector, or by increasing or decreasing the stability ofsaid vector.

The microbial cell is preferably a fungal cell, a yeast cell, or abacterial cell. Saccharomyces cerevisiae, Schizosaccharomyces pombe andPichia pastoris are preferred yeast cells and a among bacterial cellsare lactic acid bacteria preferred, particularly lactic acid bacterialspecies such as e.g. Lactococcus lactis, Lactococcus lactis subsp.lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp.diacetylactis, Leuconostoc species, Lactobacillus species, Pediococcusspecies and similar industrially relevant species like e.g.Bifidobacterium.

Also preferred are algae such as e.g. Chlamydomonas reinhardi, slimemoulds such as e.g. Dictyostelium discoideum and filamentous fungi suchas species of Neurospora and Aspergillus such as e.g. Neurospora crassaand Aspergillus nidulans, Aspergillus niger, Aspergillus crytae andPenicillium chrysogenum. Particularly preferred are also manyindustrially relevant yeast cells, slime moulds and filamentous fungiproviding a production of products such as e.g. antibiotics, steroids,pigments, enzymes, organic alcohols and acids, amino acids,polysaccharides and the like.

Among preferred microbial prokaryotes are bacterial cells such asGram-positive species such as e.g. Bacillus subtilis, Bacillusthuringensis, Bacillus licheniformis, Bacillus lentus and Bacilusstearothermophilus, as well as Gram-negative species such as Escherichiacoli. Particularly preferred are also lactic acid bacterial species suchas e.g. Lactococcus lactis, Lactococcus lactis subsp. lactis,Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp.diacetylactis, Leuconostoc species Lactobacillus species, Pediococcusspecies and similar industrially relevant species like e.g.Bifidobacterium.

In one embodiment of the method, the microbial cell is a yeast cell andthe first expressible enzyme activity is a glutamate synthase activity,preferably a glutamate synthase activity encoded by GLT1 ofSaccharomyces cerevisiae TN17 as deposited under DSM Accession Number12275, or a functionally equivalent activity. The second expressibleenzyme activity is a glutamine synthetase activity, preferably aglutamine synthetase activity encoded by GLN1 of Saccharomycescerevisiae TN15 as deposited under DSM Accession Number 12274, or afunctionally equivalent activity. The third expressible enzyme activity,when present in said cell, is a glutamate dehydrogenase activity andpreferably an activity encoded by GDH1 of Saccharomyces cerevisiae.There is also provided a method wherein said cell is Saccharomycescerevisiae TN19 as deposited under DSM Accession Number 12276. Themethod may comprise a further step of freezing or freeze-drying themicrobial cell in the preparation of a reconstitutable lyophilisate.

In one presently preferred embodiment the microbial cell is a lacticacid bacteria as exemplified herein, and said fourth expressible enzymeactivity is, at least in its native host organism, a cytoplasmictranshydrogenase, said expression of said cytoplasmic transhydrogenaseresulting in an altered and/or novel product formation and/or metaboliteproduction of said lactic acid bacteria.

An example of said novel product and/or metabolite formation is givenherein below. Lactic acid bacteria metabolise pyruvate through a numberof different pathways. The metabolite is converted into lactate bylactate dehydrogenase, into acetyl-CoA and CO₂ by pyruvatedehydrogenase, into formate by pyruvate formate lyase and intoacetolactate and CO₂ by acetolactate decarboxylase. The carbon fluxdistribution through these pathways is dependent on the external growthconditions. This control is exerted through changes in the intracellularNADH/NAD⁺ ratio (C. Garrigues, P. Loubicre, N. D. Lindley and M.Cocaign-bousquet (1997). J. Bacteriology 179, 5282–5287; F. Lopez deFelipe, M. Kleerebezem. W. M. de Vos and J. Hugenholtz (1998). J.Bacteriology 180, 3804–3808). This is illustrated by the observationsfrom physiological studies of lactic acid bacteria that are listedbelow.

Lactic acid bacteria with an increased formation of the secondarymetabolite diacetyl are relevant for a number of industrialapplications. It has been shown that overexpression of NADH-oxidase fromStreptococcus mutans in L. lactis results in a shift from homolactic(production of lactic acid) to mixed acid fermentation (production oflactic acid, acetic acid, acetoin and diacetyl) under aerobic growthconditions. This effect is ascribed to a decrease in the intracellularNADH/NAD⁺ ratio of the recombinant strain (F. Lopez de Felipe, M.Kleerebezem, W. M. de Vos and J. Hugenholtz (1998), J. Bacteriology 180,3804–3808). Expression of the cytoplasmic transhydrogenase in lacticacid bacteria is expected to have a similar effect on the NADH/NAD⁺ratio if the reaction occurs in the direction from NADH to NADPH.

It has been shown that the product formation by L. lactis changes whenthe carbon source is shifted from glucose to lactose and that thiseffect is due to a lower flux through glycolysis, resulting in a lowerNADH/NAD⁺ ratio (C. Garrigues, P. Loubiere, N. D. Lindley and M.Cocaign-bousquet (1997). J. Bacteriology 179, 5282–5287). Thus, underanaerobic growth conditions on glucose the majority (93%) of the carbonsource was converted into lactate, while only 4% of the carbon sourcewas converted into lactate when lactose was used as carbon source. Theremaining part was converted into formate, acetate and ethanol. Theauthors state that lactate formation is reduced due to a 3-fold lowerNADH/NAD⁺ ratio during growth on lactose as compared with glucose,resulting in deactivation of lactate dehydrogenase. Instead, the carbonflux towards the pyruvate node is redirected towards formation ofacetate, ethanol and formate in order to synthesise ATP and reoxidiseNADH. Expression of the cytoplasmic transhydrogenase in lactic acidbacteria is expected to have a similar effect on the NADHINAD+ ratio ifthe reaction occurs in the direction from NADH to NADPH. Thus, it isexpected that cultivation of a transhydrogenase-containing recombinantstrain of lactic acid bacteria under anaerobic growth conditins willresult in production of several new byproducts besides lactate.

Since lactate dehydrogenase is activated by a high NADH/NAD⁺ ratio it isexpected that the flux towards lactate can be increased by expressingtranshydrogenase in lactic acid bacteria under conditions where thetranshydrogenase reaction occurs in the direction from NADPH to NADH.

The invention will be further exemplified in the below provided examplesdirected to preferred embodiments of the invention. It will beunderstood that the invention is by no means limited to said examples.The examples include figures illustrating the invention and the legendsto said figures are listed below.

FIGURE LEGENDS

FIG. 1 shows plasmid pCHA 1-GDH2 integration into the GDH2 locus.

FIG. 2 shows plasmid pPGK-GDH2 integration into the GDH2 locus.

FIG. 3 shows the specific enzyme activities of the NADPH-dependent andNADH-dependent glutamate dehydrogenases in protein extracts from biomasssamples withdrawn from the continuous cultivations of strains TN1, TN9and TN12 with increasing amounts of serine in the feed. Dotted line, ●:Gdh1p activity in TN1. Normal line, ●: Gdh2p activity in TN1. Normalline, ▪: Gdh2p activity in TN9. Normal line, Δ: Gdh2p activity in TN12.

FIG. 4 shows the specific uptake rates of ammonium and serine in thecontinuous cultivations of strains TN1, TN9 and TN12 with increasingamounts of serine in the feed. Dotted lines: uptake of ammonium. Normallines: uptake of serine. ●: TN1, ▪: TN9, Δ: TN12.

FIG. 5 shows the measured specific production rates of glycerol (normallines) and the calculated specific production rates of glycerol (dottedlines) in the continuous cultivations of strains TN1, TN9 and TN12 withincreasing amounts of serine in the feed. ●: TN1, ▪: TN9, Δ: TN12.

FIG. 6 shows the specific uptake rates of glucose (dotted lines) and thespecific production rates of ethanol (normal lines) in the continuouscultivations of strains TN1, TN9 and TN12 with increasing amounts ofserine in the feed. ●: TN1, ▪: TN9, Δ: TN12.

FIG. 7 shows the consumption of glucose (▪), and production of ethanol(●), glycerol (Δ), acetate (⋄) and carbon dioxide (X) versus time in oneof the anaerobic, glucose-limited batch cultivations of strain TN1 withammonium as nitrogen source.

FIG. 8 shows the consumption of glucose (▪), and production of ethanol(●), glycerol (Δ), acetate (⋄) and carbon dioxide (X) versus time in oneof the anaerobic, glucose-limited batch cultivations of strain TN1 withserine as nitrogen source.

FIG. 9 shows plasmids pPGK-GLN1 and pPGK-GLT1 constructed in this studyand used to integrate the strong constitutive promoter of PGK into thechromosome in front of GLN 1 and GLT1, respectively.

FIG. 10 shows the natural logarithm of the biomass concentrations ofstrains TN1 (◯), TN9 (Δ), TN17 (▪) and TN19 (♦) versus time duringexponential growth in the anaerobic, glucose-limited batch cultivations.The equations show the slopes and intersections with the second axis ofthe trendlines through the measured values. The slopes are equal to themaximum specific growth rates of the strains.

FIG. 11 shows the consumption of glucose (▴) and production of ethanol(□), glycerol (♦) and carbon dioxide (X) in one of the anaerobic batchcultivations of strain TN1.

FIG. 12 shows the consumption of glucose (▴) and production of ethanol(□), glycerol (♦) and carbon dioxide (X) in one of the anaerobic batchcultivations of strain TN9.

FIG. 13 shows the consumption of glucose (▴) and production of ethanol(□), glycerol (♦) and carbon dioxide (X) in one of the anaerobic batchcultivations of strain TN19.

EXAMPLE 1 Expression of NADH-dependent and NADPH-dependent GlutamateDehydrogenase Activities in Yeast

Introduction

This example comprises a study of strains of Saccharomyces cerevisiaewith a deletion in GDH1 and a concomitant constitutive or inducibleoverexpression of GDH2. The effect on growth rates, enzyme activitiesand product formation is reported. Batch and continuous cultivations ofthe novel genetically engineered strains were carried out in highperformance bioreactors.

Materials and Methods

Microorganisms and their maintenance. All Saccharomyces cerevisiaestrains were generated from Saccharomyces cerevisiae T23D. The strainwas kindly provided by Jack Pronk from the Department of Microbiologyand Enzymology, Kluyver Laboratory of Biotechnology, Delft University ofTechnology, The Netherlands. The yeast strains were maintained at 4° C.on YPG agar plates, monthly prepared from a lyophilised stock kept at−80° C. Escherichia coli DH5α (F⁻F80dlacZ DM15 D(lacZYA-argF) U169 deoRrecA1 endA1 hsdR17(r_(k) ⁻m_(k) ⁺)supE44 i^(− thi-)1 gyra96 relA1)(GIBCOBRL, Gaithersburg, Md., USA) was used for subcloning.

Preparation of DNA. Plasmid DNA from E. coli was prepared with Qiagencolumns (Qiagen GmbH, Dûsseldorf, Germany) following the manufacturer'sinstructions. For the purification of DNA fragments used for cloningexperiments, the desired fragments were separated on 0.8% agarose gels,excised and recovered from agarose using the Qiagen DNA isolation kit(Qiagen GmbH, Dûsseldorf, Germany). Chromosomal DNA from Saccharomycescerevisiae was extracted as follows. Cells were grown in medium in shakeflasks and harvested at OD=1.5, 10 mg of wet cells were resuspended in0.5 ml Tris-Cl (pH 8.0) and quenched with 0.5 ml glass beads (size250–500 microns) in the presence of 0.5 ml Tris-saturated phenol (pH8.0). The DNA was extracted from the phenol phase with chloroform,precipitated with 98% ethanol and resuspended in TE buffer. RNA in theextract was removed by treatment with RNAaseA (purchased from Promega)and finally the DNA was purified by precipitation with ethanol/lithiumchloride and resuspended in TE buffer. The DNA primers were purchasedfrom DNA Technology (Aarhus, Denmark).

Deletion of GDH1. Plasmid pGDH1del was kindly donated by professor F. K.Zimmermann (Boles et al., 1993). In pGDH1del a 1.0 kb fragment of GDH1has been replaced by a 11.1 kb fragment containing the open readingframe of URA3. The construct was linearised with ClaI/PvuII prior totransformation. Correct deletion of GDH1 was verified by PCR analysisand by measurements of GDH1p activity in protein extracts fromtransformants. No NADPH-dependent glutamate dehydrogenase activity couldbe detected in correct transformants.

Overexpression of GDH2. The CHA1 promoter was cloned by PCR using pfupolymerase (New England Biolabs) and the primers CHA1 start (5′-ATT CATCGA TGA ATT CTA TCT TAT GGT CCC ATT CTT TAC TGC ACT GTT TAC A-3′), SEQID NO:3, consisting of restriction enzyme sites for ClaI and EcoRI infront of nucleotides -364 to -329 uptream of CHA1) and CHA1 stop (5′-GGCCAC TAG TGA TAT CAA AGC ATT CTC TCG CTG GTT AAT TTT CCT GTC TCT TGT CTATCA GCA CTT AAA AA-3′), SEQ ID NO:4, consisting of restriction enzymesites for SpeI, EcoRV and BsmI in front of nucleotides -1 to -45upstream of CHA1). The resulting DNA fragment was isolated after gelelectrophoresis on a 0.8% agarose gel and subcloned into the SmaI sitein vector pUC19, resulting in plasmid pCHA1. The CHA1 promoter wasisolated by from pCHA1 by digestion with BsmI and HineII (located in themulti-cloning site of pUC19). Plasmid YEpMSP3, containing the openreading frame of GDH2 (Boles et al., 1993), was kindly donated byProfessor F. K. Zimmermann. The plasmid was digested with MscI and BsmIand ligated with the HincII/BsmI CHA1p fragment, resulting in a plasmidwith insertion of the CHA1 promoter in front of the GDH2 start codon.This construct was digested with BamHI and a fragment, consisting of theCHA1 promoter and 2.27 kb of the open reading frame of GDH2, wasisolated. Plasmid pFA6A-kanMX3 contains the geniticin resistance gene,G418′, flanked by two direct repeats and two multi-cloning sites (Wachet al., 1994). A EcoRI/MscI DNA fragment, consisting of nucleotides -500to -136 upstream of GDH2, was isolated from YEpMSP3 and inserted intopFA6A-kanMX3, digested with EcoRI and EcoRV. The resulting construct waslinearised by digestion with BamHI and ligated with the 2.64 kbCHA1p-GDH2 fragment, resulting in plasmid pCHA1CGDH2 (FIG. 1). pCHA1GDH2was linearised with SpeI/AatII prior to transformation. It was verifiedby PCR that the CHA1 promoter was inserted in front of the open readingframe of GDH2 on chromosome IV of correct transformants. For thispurpose primers CHA1start and GDH2verif (5′-GGT TTT CTA CAA TCT CCA AAAGAG-3′), SEQ ID NO:5, spanning the region from nucleotides 1294 to 1271of the GDH2 open reading frame.

Primers Gdh2start (5′-GCG CGA GAT CTT CTA GAA TGC TTT TTG ATA ACA AAAAT-3′), SEQ ID NO:6, containing restriction enzyme sites for BglII andXbaI in front of nucleotides 1 to 21 of GDH2, and Gdh2stop (5′-CGC GCAGAT CTC CGC GGA GAG CCT AAA CGA TTA ACA AA-3′), SEQ ID NO:7, containingrestriction enzyme sites for BglII and SacII in front of nucleotides1221 to 1201 of GDH2, were used to clone parts of the structural gene ofGDH2 by PCR with pfu polymerase (New England Biolabs). A DNA fragment ofthe correct size was isolated from a 0.8% agarose gel afterelectrophoresis and digested with BglII overnight. The fragment wasligated into the unique BglII digestion site of plasmid Yep24-pPGKbehind the PGK promoter and in front of the PGK terminator (Walfridssonet al., 1997), resulting in plasmid Yep24-pPGK-GDH2. A 2.65 kbSmaI/SacII DNA fragment, consisting of the PGK promoter and the clonedpart of GDH2 was isolated from Yep24-pPGK-GLT1. The fragment was ligatedinto plasmid pFA6A-kanMX3 (Wach et al., 1994), digested with EcoRV andSacII, resulting in plasmid pPGK-GDH2 (FIG. 2). The plasmid waslinearised by digestion with TthIII1 prior to transformation. Correctinsertion of the plasmid into the GDH2 locus on chromosome IV wasverified by PCR analysis of chromosomal DNA extracted from transformantswith resistance towards geniticin. For this purpose primers PGKverif(5′-GTC ACA CAA CAA GGT CCT A-3′), SEQ ID NO:8, spanning the region fromnucleotides -420 to -400 upstream of the PGK start codon, and Gdh2verif(described above) were used.

Transformation of E. coli and S. cerevisiae. E. coli DH5α wastransformed by electro-transformation using the Bio-Rad electroporationequipment (Biorad Laboratories. Richmond, USA). Transformants wereselected on L broth plates containing 100 mg/ml ampicillin. S.cerevisiae cells were made competent for plasmid uptake by treatmentwith lithium acetate and polyethyleneglycol (Schiestl & Gietz, 1989). 5μg of DNA was used for each transformation. Transformants were plateddirectly on selective media except for the G418 resistant transformants.These were suspended in YPD for 24 hours prior to plating on selectivemedia in order to obtain expression of the G418 resistance gene. Correctintegration of the fragments from pHOde1 and pSUC2 into the chromosomewas verified by PCR analysis using extracted DNA from the transformants.

Medium in the batch and continuous cultivations. The strains of S.cerevisiae were cultivated in a mineral medium prepared according toVerduyn et al. (1990). Vitamins were added by sterile filtrationfollowing heat sterilisation of the medium. The concentrations ofglucose and (NH₄)₂SO₄ initially in the batch cultivations were 25 g perl and 3.75 g per l, respectively. The concentration of glucose in thefeed to the continuous cultivations was 25 g per l while theconcentration of (NH₄)₂SO₄ was varied from 3.75 g per l to 0 g per l.The serine concentration in the feed was varied from 0 g per l to 3.16 gper l so that the sum of the ammonium and serine concentrations was 60mM in all cultivations. Growth of S. cerevisiae under anaerobicconditions requires the supplementary addition to the medium ofergosterol and unsaturated fatty acids, typically in the form of Tween80 (Andreasen & Stier, 1953; Libudzisz et al. 1986). Ergosterol andTween 80 were dissolved in 96% (v/v) ethanol and the solution wasautoclaved at 121° C. for 5 min. The final concentrations of ergosteroland Tween 80 in the medium were 4.2 mg per g DW and 175 mg per g DW,respectively. To prevent foaming 75 μl per l antifoam (Sigma A-5551) wasadded to the medium. The medium reservoir for the continuouscultivations was extensively sparged with N₂ containing less than 5 ppmO₂ after preparation and was then sealed. To avoid formation of a vacuumwhen withdrawing medium form the reservoir it was connected to a gasimpermeable bag filled with N₂ containing less than 5 ppm O₂.

Experimental set-up for the batch and continuous cultivations. Anaerobicbatch and continuous cultivations were performed at 30° C. and at astirring speed of 800 rpm in in-house manufactured bioreactors. Theworking volume of the batch reactors and the continuous cultivationreactors were 4.5 liters and 1.0 liters, respectively. pH was keptconstant at 5.00 by addition of 2 M KOH. The bioreactors were equippedwith off-gas condensers cooled to 2° C. The bioreactors werecontinuously sparged with N₂ containing less than 5 ppm O₂, obtained bypassing N₂ of a technical quality (AGA 3.8), containing less than 100ppm 0, through a column (250×30 mm) filled with copper flakes and heatedto 400° C. The column was regenerated daily by sparging it with H₂ (AGA3.6). A mass flow controller (Bronkhorst HiTec F201C) was used to keepthe gas flow into the bioreactors constant at 0.50 l nitrogen per minper liter Norprene tubing (Cole-Parmer Instruments) was used throughoutin order to minimise diffusion of oxygen into the bioreactors. Thebioreactors were inoculated to an initial biomass concentration of 1 mgper l with precultures grown in unbaffled shake flasks at 30° C. and 100rpm for 24 hours. The anaerobic batch cultivations of strains TN1, TN9,TN12 and TN22 were each carried out three times with identical results.Steady state in the continuous cultivations was obtained after growthfor 10–11 residence times. This was verified by measuring a constantformation of CO₂ and medium components, e.g. ethanol, glycerol andacetate, by the yeast throughout 2–3 residence times.

Determination of dry weight. Dry weight was determined gravimetricallyusing nitrocellulose filters (pore size 0.45 μm; Gelman Sciences). Thefilters were predried in a microwave oven (Moulinex FM B 935Q) for 10min. A known volume of culture liquid was filtered and the filter waswashed with an equal volume of demineralised water followed by drying ina microwave oven for 15 min. The relative standard deviation (RSD) ofthe determinations was less than 1.5% based on triple determinations(n=3).

Analysis of medium compounds. Cell-free samples were withdrawn directlyfrom the bioreactor through a capillary connected to a 0.45 μm filter.Samples were subsequently stored at −40° C. Glucose, ethanol, glycerol,acetic acid, pyruvic acid, succinic acid and 2-oxoglutarate weredetermined by HPLC using an HPX-87H Aminex ion exclusion column(RSD<0.6%, n=3). The column was eluted at 60° C. with 5 mM H₂SO₄ at aflow rate of 0.6 ml per min. Pyruvic acid, acetic acid and2-oxoglutarate were determined with a Waters 486 UV meter at 210 nmwhereas the other compounds were determined with a Waters 410 refractiveindex detector. The two detectors were connected in series with the UVdetector first. The CO₂ concentration in the off-gas was determinedusing a Brüel & Kjær 1308 acoustic gas analyser (RSD=0.02%) (Christensenet al, 1995). Ammonium was determined using a commercially availableassay (Boehringer Mannheim Cat. No. 1 112 732). Serine was determined asdescribed by Barkholt and Jensen, 1989. In a separate experiment the offgas from the bioreactor was bubbled through liquid nitrogen and theethanol concentration in the frozen mixture of water, ethanol andacetaldehyde was determined by HPLC after evaporation of the N₂. Herebythe loss of ethanol through the reflux condenser of the bioreactor wasdetermined to be between 4% and 9% of the ethanol formed by thebioreaction depending on the dilution rate (Schulze, 1995). In thecarbon balances the measured ethanol fluxes were corrected for this lossthrough evaporation.

Measurement of enzyme activities. Culture liquid was withdrawn from thebioreactor into an ice cooled beaker, centrifuged and washed twice with10 mM potassium phosphate buffer (pH 7.5, 2° C.) containing 2 mM EDTA.Subsequently the cells were resuspended in 4.2 ml 100 mM potassiumphosphate buffer (pH 7.5, 2° C.) containing 2 mM MgCl₂ followed byimmediate freezing in liquid nitrogen and storage at −40° C. Prior toanalysis 0.22 ml of 20 mM DTT was added to the samples whereafter theywere distributed into precooled 2 ml eppendorf tubes containing 0.75 mlglass beads (size 0.25–0.50). The cells were disrupted in a bead millfor 12.5 min. (0° C.). The test tubes were centrifuged (20000 rpm, 20min., 0° C.) whereafter the supernatants were pooled in one test tube.During the following analyses the extract was kept on ice. Enzyme assayswere performed at 30° C. using a Shimadzu UV-260 spectrophotometer at30° C. Reaction rates, corrected for endogenous rates, were proportionalto the amount of extract added. All enzyme activities are expressed asmicromole of substrate converted per minute per mg total cellularprotein as determined by the Lowry method. Glutamate dehydrogenase (NAD⁺and NADP⁺) (EC 1.4.1.5 and EC 1.4.1.4, respectively) were assayed asdescribed by Bruinenberg et al. (1983a). Glutamate synthase (GOGAT) (EC1.4.1.14) was assayed as described by Holmes et al. (1989).

Results

Construction of strains with a deletion in GDH1 and an overexpression ofGDH2. The object of the study was to analyse whether the NADH-dependentglutamate dehydrogenase, encoded by GDH2, could substitute theNADPH-dependent isoenzyme, encoded by GDH1, in assimilation of ammoniumand 2-oxoglutarate into glutamate in S. cerevisiae. This should lead toa reduction in surplus formation of NADH in biomass synthesis and thus,to a S. cerevisiae strain with a reduced formation of glycerol andpossibly an increased formation of ethanol. To obtain this, strain wereconstructed with a deletion in GDH1 and an overexpression of GDH2. GDH1was deleted as described earlier (Boles et al., 1993) in the haploidstrain TN2 derived from S. cerevisiae CBS8066 (Nissen et al., 1998). Theresulting strain was denounced TN9. In order to obtain a strain with astable, constitutive overexpression of GDH2, the promoter of PGK,encoding phosphoglycerate kinase, was inserted in front of the startcodon of GDH2 on chromosome IV. PGK encodes one of the most abundantmRNA and protein species in the cell, accounting for 1% to 5% of thetotal cellular mRNA and protein during growth on fermentative carbonsources (Dobson et al. 1982). Insertion of the promoter was obtained byhomologue recombination of a 4.8 kbp SpeI/xxx fragment from pPGKGDH2into the GDH2 locus in strain TN9 (see materials and methods). Theresulting strain was denounced TN22. The promoter of CHA1, encoding thecatabolic L-serine dehydratase, has been reported to be inducible by lowamounts of serine (Bornæs et al., 1993). To study to effect of varyinglevels of Gdh2p activity in a strain background with no activity ofGdh1p, the CHA1 promoter was inserted into chromosome IV in front of theopen reading frame of GDH2 in strain TN9. The serine inducible promoterwas chosen to avoid the use of inducible promoters that were dependenton addition of a second carbon source besides glucose since this wouldcomplicate the comparison between cultivations with increasingconcentrations of this second carbon source. Furthermore, the CHA1promoter was reported to be induced up to 130 times by addition of 5 mMserine, which would have very little influence on the cell physiology.The insertion of the CHA1 promoter was obtained by homologuerecombination of a 4.3 kbp SpeI/AatII fragment from pCHA1GDH2 into theGDH2 locus in strain TN9 (see materials an methods). The resultingstrain was denounced TN12.

Continuous cultivations. Physiological studies of the geneticallyengineered strains were carried out in anaerobic continuouscultivations. Strain TN12 with the inducible CHA1 promoter inserted intothe chromosome in front of GDH2 was cultivated until steady states hadbeen achieved in growth media containing glucose as the primary carbonsource and increasing amounts of serine from 0 mM to 30 mM. The amountof ammonium sulphate in the feed was regulated to give a finalconcentration of 60 mM ammonium and serine. This was done to study thedegree of GDH2 induction that could be achieved with the new promoterand the effect of this induction on product formation. Similarcultivations of strains TN1 and TN9 were performed. Hereby, the effectsof the increasing amounts of serine in the feed, the deletion of GDH1,and the insertion of the new promoter in front of GDH2 on the productformation could be discriminated from each other.

The specific activities of Gdh1p, Gdh2p and Glt1p were measured in vitroin cell extracts from each steady-state cultivation. The activity ofGdh1p in TN1 decreased almost linear from 0.657 to 0.475 units per mgtotal cellular protein (Upper mg TCP) when the serine content in thefeed was increased from 0 mM to 30 mM (dotted line in FIG. 3). Thisfitted well with the measured decrease in ammonium uptake from 18.8 to8.2 mmoles per c-mole biomass per hour, respectively. Earlier studies ofS. cerevisiae have shown that a decrease in ammonium uptake results in asimultaneous reduction in Gdh1p activity in cell free extracts (terSchure et al., 1995a). No activity of the NADPH-dependent glutamatedehydrogenase could be detected in cell free extracts from any of thecontinuous cultivations of TN9 and TN12. The Gdh2p activity in cell freeextracts of strain TN1 increased from 0.007 Upper mg TCP when ammoniumwas used as the sole nitrogen source to 0.022 Upper mg TCP when 30 mMserine was present in the feed (FIG. 3). Deletion of GDH1 in TN9resulted in a marked increase in the specific activity of Gdh2p. Whenammonium was used as the sole nitrogen source the activity wasdetermined to 0.268 Upper mg TCP, an increase of a factor 40 as comparedto the activity in TN1. It has been shown that the intracellular levelof glutamine results in strong repression of GDH2 expression at thetranscriptional level (Miller and Magasanik, 1991). When GDH1 is deletedthe synthesis of glutamate from 2-oxoglutarate and ammonium is stronglyaffected which probably leads to a significant decrease in theintracellular concentration of the component. Since glutamate is one ofthe substrates in glutamine synthesis this will result in a furtherdecrease in the intracellular level of the amino acid. Thus, the strongincrease in Gdh2p in TN9 probably was due to an elevation of glutaminerepression of GDH2. Addition of increasing amounts of serine to the feedresulted in a significant reduction in the Gdh2p activity. Since theresidual glucose concentrations in all cultivations of TN9 was constantat 20 c-mmoles per liter this was not due to glucose repression of GDH2(Coschigano et al., 1991). The residual concentration of ammoniumdecreased from 48 mM when the compound was used as the sole nitrogensource to 28 mM when 30 mM serine was added to the medium. In an earlierstudy in continuous cultivations of a wild-type S. cerevisiae it wasdemonstrated that the Gdh2p activity decreased by a factor 4 when theresidual ammonium concentration was reduced by this amount (ter Schureet al., 1995b). Since the intracellular glutamine concentrationdecreased simultaneously, this effect was not due to repression fromthis component. Instead, an observed increase in the intracellularconcentration of 2-oxoglutarate might explain the reduction in Gdh2pactivity since this component generally is thought to be the product ofthe reaction catalysed by the enzyme when the cells are cultivated onnitrogen sources other than ammonium. No increase in the specificactivity of Gdh2p was observed in the cultivation of TN12 with ammoniumas the sole nitrogen source. This clearly demonstrated that the increaseof Gdh2p activity observed under these growth conditions in TN9 was dueto deregulation of the GDH2 promoter. The expected induction of the CHA1promoter by serine was not observed in the cultivations of TN12 whenincreasing amounts of the component was added to the feed. The presenceof 5 mM serine in the medium increased the Gdh2p activity by a factor of6 while the maximum induction was observed by addition of 30 mM serine.Here the activity of the enzyme was 13 times higher than when ammoniumwas used as nitrogen source. No mutations in the cloned region of theCHA1 promoter was found when the obtained PCR fragment was sequenced. Asmentioned above earlier studies have shown that the promoter is induced130 times at the transcriptional level by the presence of 5 mM serine inthe growth medium. Thus, the absence of this induction in TN12 must bedue to post-transcriptional regulation of the enzyme. No significantdifference in the specific activity of glutamate dehydrogenase could bedetected when steady states cultivations with increasing amounts ofserine in the feed of the same strain were compared or when steady statecultivations of TN1, TN9 and TN12 were compared. The level of thespecific activity ranged from 0.008–0.013 Upper mg TCP.

The uptake of glucose, ammonium and serine and the production ofethanol, glycerol, biomass, carbon dioxide, succinate, pyruvate andacetate by the three strains were measured at each steady state. Fromthese measurements the product yields (in c-moles product produced perc-moles glucose and serine consumed) were calculated (Tables 1–3).

TABLE 1 Ammonium in the feed, mM 60 55 45 30 Serine in the feed, mM 0 515 30 Ethanol 0.491 0.503 0.518 0.523 Glycerol 0.078 0.067 0.058 0.050Pyruvate 0.003 0.001 0.002 0.002 Acetate 0.001 0.003 0.001 0.002Succinate 0.003 0.003 0.003 0.004 Carbon dioxide 0.239 0.232 0.233 0.248Biomass 0.141 0.131 0.137 0.152 Total 0.956 0.940 0.952 0.981 Table 1.Product yields in the continuous cultivations of strain TN1 withincreasing amounts of serine in the feed. Unit: c-moles product perc-moles of glucose and serine consumed.

TABLE 2 Ammonium in the feed, mM 60 55 45 30 Serine in the feed, mM 0 515 30 Ethanol 0.525 0.532 0.550 0.572 Glycerol 0.062 0.044 0.028 0.012Pyruvate 0.004 0.003 0.002 0.003 Acetate 0.000 0.000 0.000 0.000Succinate 0.003 0.004 0.003 0.004 Carbon dioxide 0.257 0.267 0.274 0.274Biomass 0.123 0.122 0.124 0.106 Total 0.974 0.972 0.981 0.971 Table 2.Product yields in the continuous cultivations of strain TN9 withincreasing amounts of serine in the feed. Unit: c-moles product perc-moles of glucose and serine consumed.

TABLE 3 Strain TN12 TN22 Ammonium in the feed, 60 55 45 30 60 mM Serinein the feed, mM 0 5 15 30 0 Ethanol 0.500 0.529 0.534 0.546 0.516Glycerol 0.061 0.045 0.026 0.014 0.030 Pyruvate 0.002 0.002 0.001 0.0010.002 Acetate 0.004 0.004 0.004 0.004 0.004 Succinate 0.003 0.003 0.0030.004 0.004 Carbon dioxide 0.247 0.279 0.250 0.265 0.255 Biomass 0.1250.128 0.130 0.138 0.138 Total 0.942 0.990 0.948 0.972 0.949 Table 3.Product yields in the continuous cultivations of strain TN12 withincreasing amounts of serine in the feed and in the continuouscultivation of strain TN22 with ammonium as the sole nitrogen source.Unit in the cultivations of TN12: c-moles product per c-moles of glucoseand serine consumed. Unit in the cultivation of TN22: c-moles productper c-mole glucose consumed.

Carbon balances of the products yields in the 12 steady statecultivations showed that 95%–98% of the consumed carbon was convertedinto one of the listed products. The degree of reduction of theremaining 2%–5% of the consumed carbon varied between 0 and 1,indicating that the production of carbon dioxide was measured to low.

Ammonium and serine were consumed simultaneously by the three strains(FIG. 3). Thus, the presence of both nitrogen sources in the growthmedium did not result in repression of the specific uptake systems ofthe two components. The increasing amounts of serine added to the feedresulted in an increase in the specific uptake of the compound. A modelbased on simple Michaelis Menten type kinetics, describing thedependence of the uptake on the extracellular serine concentration, didnot fit the measurements (results not shown). Thus, other components,e.g. glucose, probably had a regulatory effect on the specific uptake ofthe compound. The need for nitrogen uptake in the form of ammoniumdecreased when increasing amounts of serine were consumed by the cells.This resulted in a reduction in the specific ammonium uptake in thecultivations (FIG. 4). The total specific uptake of nitrogen wascalculated from the measurements of the ammonium and serine uptake to beconstant at 17–18 mmoles nitrogen per c-mole biomass per hour in all 12steady state continuous cultivations. This indicated a constantcomposition of the biomass with respect to the content of protein, RNA,DNA and carbohydrates.

Addition of serine to the medium resulted in a decrease in the specificglycerol production (FIG. 5). The serine addition reduces the cells needfor de novo synthesis of the amino acid. Synthesis of one mole serinefrom glucose and ammonium involves the reduction of two moles NAD⁺ toNADH and a deamination step where one mole glutamate is converted to2-oxoglutarate. As described earlier glutamate can be regenerated from2-oxoglutarate by either two isoenzymes of glutamate dehydrogenase,encoded by GDH1 and GDH2, under consumption of NADPH or NADH,respectively. Furthermore, glutamate can be synthesised from2-oxoglutarate and glutamine by glutamate synthase, encoded by GLT1,under consumption of NADH (Cogoni et al., 1995). In strain TN1 all threeenzymes are present while only Gdh2p and Glt1p are active in strains TN9and TN12. Thus, addition of serine results in a reduction of surplusNADH formation of either 1 or 2 moles per mole serine in TN1 and 1 moleper mole serine in TN9 and TN12. De novo synthesis of serine in S.cerevisiae CBS8066 was calculated to 157 c-mmoles per c-mol biomassbased on measurements of the content of serine and the amino acidssynthesised from serine in the protein pool (Schulze, 1995). By assumingthis to be similar in TN1, TN9 and TN12 the specific serine synthesisduring growth on ammonium as the sole nitrogen source was 19.9 c-mmolesper c-mole biomass per hour. In the cultivations with 15 mM and 30 mMserine in the feed the specific serine uptake exceeded this value (FIG.4). Thus, in these cultivations serine was also catabolised by thecells. As mentioned above L-serine dehydratase, encoded by CHA1,catalyses deamination of serine to pyruvate and ammonium. No increase inthe secretion of pyruvate, acetate or metabolites in the tricarboxyliccycle was measured when increasing amounts of serine was added to thefeed. Hence, pyruvate stemming from the deamination reaction wasconverted into acetaldehyde and further into ethanol under consumptionof one mole NADH per mole serine. These two effects of serine additionto the medium on the reduction of surplus NADH formation resulted in theobserved decrease in the specific glycerol production. This wasquantified by extracting the reduction in surplus NADH formation due toserine addition from the value of the specific glycerol productionmeasured in the continuous cultivations with ammonium as the solenitrogen source (dotted lines in FIG. 5). In all calculations a valuefor the reduction in surplus NADH formation of 1 mole per mole of serinewas used. The reduction in the specific glycerol formation of strainsTN9 and TN12 when cultivated in media with increasing amounts of serinein the feed could be accounted for by the two effects of serine additionon surplus NADH formation since the calculated values were close toequal with the measured values. For strain TN1 the calculated specificglycerol production was equal to the measured values in the cultivationswith 15 mM and 30 mM serine in the feed. In the cultivation with 5 mMserine in the feed a correct value for the specific glycerol formationcould only be calculated by assuming a reduction in surplus NADHformation of 2 moles per mole serine. Hereby a specific glycerolproduction of 61.2 c-mmoles per c-mole biomass per hour was obtained ascompared to 68.6 c-mmoles per c-mole biomass per hour when a value forthe reduction in surplus NADH formation of 1 mole per mole of serine wasused. This indicates that Gdh1p catalyses the regeneration of glutamatefrom 2-oxoglutarate when the serine uptake is low while Gdh2p catalysesthe reaction with increasing levels of the amino acid. Earlier studiessupport this observation since it has been shown that Gdh1p catalysesthe formation of glutamate during growth on ammonium as the solenitrogen source while Gdh2p catalyses the interconversion of glutamateand 2-oxoglutarate during growth on other nitrogen sources (Courchesneand Magasanik 1988: Miller and Magasanik, 1990). The calculationsclearly demonstrated the significant effect of serine addition on thecell physiology but also that formation of surplus NADH in the synthesisof biomass has a high control on glycerol formation. Furthermore, theydemonstrated that the decrease in the specific glycerol production wasindependent of the specific activity of Gdh2p.

The specific ethanol production was also affected by addition of serineto the feed (FIG. 6). In the cultivations of TN1 the specific ethanolformation reached a maximum when 5 mM and 15 mM serine was added to thefeed and then decreased slightly when the serine content was increasedto 30 mM. In the cultivations of TN9 the specific ethanol productionincreased almost linearly from 540 to 626 c-mmoles per c-mole biomassper hour while it was constant in the cultivations of TN12. Serineaddition has at least four potential influences on ethanol formation inS. cerevisiae. The cellular consumption of ATP is to a given extentaffected by the increase in active uptake of serine, by the decrease inactive uptake of ammonium and by the reduction in glycerol formationsince glycerol synthesis involves consumption of one ATP per molecule.Since ATP is formed through synthesis of ethanol during anaerobic growththese effects will change the specific ethanol formation. Furthermore,as described above serine was catabolised to ethanol in some of thecultivations. The effects of serine addition on ethanol synthesis werequantified by assuming ATP consumption in the active uptake systems ofserine and ammonium of one ATP per molecule. The calculated changes inethanol formation due to the four effects was then used to calculatevalues for the specific ethanol formation with increasing amounts ofserine in the feed from the value measured when ammonium was used as thesole nitrogen source. An increase in the specific ethanol productionfrom 540 to 544 c-mmoles per c-mole biomass per hour and from 531 to 536c-moles per c-mole biomass per hour was calculated for strains TN9 andTN12, respectively. For TN1 the calculated specific ethanol productiondecreased from 441 to 435 c-mmoles per c-mole biomass per hour. Thus,the listed direct effects of serine addition on ATP consumption andethanol synthesis could not explain the changes in the specific ethanolproduction. Instead, the changes could be due to a switch in themetabolism leading to an increased ATP consumption in biomass synthesis.It has been shown in an earlier study that only glutamate synthasecatalyses the formation of glutamate when GDH1 is deleted (Miller andMagasanik, 1990). Thus, a strain with a double deletion in GDH1 and GLT1grew poorly on ammonium as the sole nitrogen source. In the study agrowth medium containing high levels of glucose was used and hence,Gdh2p activity in the double mutant was repressed. By overexpressingGDH2 in the mutant a specific activity of the gene product of 0.195units per mg TCP was obtained which led to an increase in the growthrate on ammonium to wild-type levels. In the continuous cultivation ofstrain TN9, carrying the deletion in GDH1, with ammonium as the solenitrogen source the specific Gdh2p activity was 0.268 units per mg TCPdue to absence of glucose repression. This strongly indicated thatglutamate was synthesised by both glutamate synthase and theNADH-dependent glutamate dehydrogenase under these growth conditions.When the serine content in the medium was increased the specific Gdh2pactivity decreased. Since the specific growth rate was kept constant at0.127 h⁻¹ it is reasonable to assume that also the cellular glutamatesynthesis rate was constant with increasing amounts of serine in thefeed. Thus, increasing amounts of the component probably was synthesisedthrough the reaction catalysed by glutamate synthase. Here2-oxoglutarate and glutamine is converted into two molecules ofglutamate under consumption of NADH. Glutamine is then regenerated fromglutamate under consumption of ammonium and ATP catalysed by glutaminesynthetase. In the net reaction catalysed by the two enzymes2-oxoglutarate and ammonium are converted into glutamate underconsumption of NADH and ATP which results in an increase in ATPconsumption in biomass synthesis compared to glutamate synthesiscatalysed by Gdh2p. Overall, the increase in the specific ethanolformation in strain TN9 with increasing amounts of serine in the feedmight be due to this increase in ATP consumption in the synthesis ofglutamate. In the cultivation of TN12 with ammonium as the sole nitrogensource glutamate synthase catalysed the synthesis of glutamate since thespecific Gdh2p activity was measured to 0.005 units per mg TCP. Asdescribed earlier the specific activity of the NADH-dependent glutamatedehydrogenase increased with increasing amounts of serine in the feedbut the level did not exceed 0.065 units per mg TCP which probably wasto low to substitute glutamate synthase in formation of glutamate.Hence, ATP consumption in biomass synthesis remained constant in thecultivations, which resulted in a constant specific ethanol formation.The NADPH-dependent glutamate dehydrogenase catalysed glutamateformation in the cultivations of TN1. Thus, an increase in ATPconsumption in the assimilation of ammonium could not explain the smallincrease in the specific ethanol formation observed in the cultivationswith 5 mM and 15 mM serine in the feed. Instead, the observed increaseof approximately 6% compared to the cultivation with ammonium as thesole nitrogen source could be due to the sum of small changes in themetabolism, resulting in an increased ATP consumption in biomasssynthesis. In the cultivation of TN9 with ammonium as the sole nitrogensource an increase in the specific ethanol production and a decrease inthe specific glycerol production of 22% and 20%, respectively, wasobserved compared to the cultivation of TN1. Similar results wereobtained in the cultivation of TN12. Thus, deletion of GDH1, resultingin formation of cellular glutamate by Glt1p, led to a S. cerevisiaestrain with improved characteristics for ethanol production.

To further analyse the effect of GDH2 overexpression in a Δgdh1 mutantwithout the influence of serine addition a glucose-limited continuouscultivation of strain TN22 was carried out with ammonium as the solenitrogen source at a dilution rate of 0.11 h⁻¹. As mentioned above TN22has the strong, constitutive promoter of PGK inserted in front of GDH2on chromosome IV. A specific activity of 0.381 units per mg TCP of theNADH-dependent glutamate dehydrogenase was measured in cell-freeextracts from the steady state cultivation of TN22 while the activitiesof Gdhp1 and Glt1p was absent and 0.012 units per mg TCP, respectively.The carbon balance and the balance over the degree of reduction of thesubstrates and products measured in the cultivation both closed within3.8%. Thus, it was concluded that all products secreted by the cell hadbeen quantified correctly. The high Gdh2p activity was reflected in themeasured specific production rates of ethanol and glycerol. The tworates were measured to 411 c-mmoles per c-mole biomass per hour and 24c-mmoles per c-mole biomass per hour, respectively. The low value forthe specific ethanol production was similar to the value measured in thecontinuous cultivation of TN1 with ammonium as the sole nitrogen source.Hence, the level of ATP consumption in the assimilation of ammonium intoglutamate probably was identical in the two strains. This indicated thatin TN22 the NADH-dependent glutamate dehydrogenase completely hadsubstituted the role of glutamate synthase in glutamate synthesis thatwas observed in strains TN9 and TN12. The very low specific glycerolproduction in TN22 compared to strain TN9 and TN12 was surprising. Thespecific uptake of ammonium in the cultivation was 19.2 mmoles perc-mole biomass per hour which was 12% higher than in TN9. Also thebiomass yield increased from 123 c-mmoles per c-mole glucose in TN9 to138 c-mmoles per c-mole glucose in TN22. This indicated that the highGdh2p activity resulted in a higher flux from 2-oxoglutarate andammonium to glutamate in TN22 than in TN9, which led to an increase inNADH reoxidation through the reaction catalysed by the enzyme, and thus,a decrease in formation of glycerol. Furthermore, it indicated that thereduction in biomass Yields observed in the continuous cultivations ofTN9 and TN12 compared to TN1 and TN22 (Tables 1–3) was due to alimitation in the ammonium assimilation. No changes in formation of theorganic acids were observed compared to TN9 and TN12.

Batch cultivations. The anaerobic physiology of the geneticallyengineered S. cerevisiae strains were also studied in batch cultivationswith glucose as the primary carbon source and either ammonium or serineas nitrogen source. This was done to quantify the effect of the geneticchanges on the maximum specific growth rate, μ_(max), and on the productyields.

The product yields obtained in the anaerobic batch cultivations of thefour strains are listed in Table 4.

TABLE 4 Strain TN1 TN9 TN12 TN22 Serine (mM) 0 60 0 60 0 60 0 Ammonium(mM) 60 0 60 0 60 0 60 Ethanol 0.480 0.491 0.520 0.534 0.526 0.531 0.500Glycerol 0.097 0.067 0.060 0.037 0.066 0.040 0.067 Pyruvate 0.003 0.0020.003 0.003 0.002 0.002 0.004 Acetate 0.003 0.028 0.004 0.018 0.0190.022 0.001 Succinate 0.003 0.003 0.002 0.004 0.003 0.004 0.003 Carbondioxide 0.261 0.262 0.275 0.278 0.272 0.275 0.262 Biomass 0.121 0.1190.114 0.105 0.072 0.107 0.126 Total 0.968 0.972 0.978 0.979 0.960 0.9810.963 μ_(max) (h⁻¹) 0.41 0.31 0.22 0.20 0.17 0.24 0.39 Table 4. Productyields in the anaerobic, glucose-limited batch cultivations of strainsTN1, TN9, TN12 and TN12 with either ammonium or serine as nitrogensource. Unit in the cultivation with ammonium as nitrogen source:c-moles product per c-mole glucose consumed. Unit in the cultivationwith serine as nitrogen source: c-moles product per c-moles glucose andserine consumed.

The glycerol yield decreased significantly when strain TN1 wascultivated with serine as nitrogen source as compared to the cultivationon ammonium. As for the continuous cultivations this was due to areduction in surplus formation of NADH in biomass synthesis under thesegrowth conditions. A dramatic increase in formation of acetate wasobserved and also the ethanol yield increased. The total cellular uptakeof serine was 309 c-mmoles per c-mole biomass. By assuming the constantneed for de novo serine synthesis of 157 c-mmoles per c-mole biomassused in the earlier calculations it could be calculated that 152c-mmoles serine per c-mole biomass was catabolised to ethanol andacetate. The specific ethanol and acetate formation increased with 92c-mmoles per c-mole biomass and 210 c-mmoles per c-mole biomass,respectively, when serine was used as nitrogen source instead ofammonium. Thus, degradation of serine to acetate could not account forthe increase in the yield of the component alone. Formation of acetateby the NAD(P)⁺-dependent aldehyde dehydrogenase, encoded by ALD2, hasbeen proposed to have a physiological role in reduction of NADP⁺ toNADPH, which is consumed in biomass synthesis (Bruinenberg et al.,1983b; Miralles and Serano, 1995). Serine is a precursor in synthesis ofcysteine and phospholipids, which are synthesis pathways that requirelarge amounts of NADPH. The presence of high concentrations ofintracellular serine could potentially increase the flux through thesepathways and account for the increase in NADPH formation throughsynthesis of acetate. The maximum specific growth rate of TN1 decreasedsignificantly from 0.41 h⁻¹ to 0.31 h⁻¹ when the nitrogen source wasswitched from ammonium to serine. When serine is used as nitrogen sourcethe nitrogen has to be made available for biomass synthesis throughdegradation of the amino acid to pyruvate and ammonium. This process isless efficient than direct uptake of ammonium followed by assimilationinto glutamate catalysed by glutamate dehydrogenase and glutamatesynthase and hence, the reduction in the specific growth rate probablywas due to this difference in nitrogen assimilation. The production ofethanol, glycerol, acetate and carbon dioxide as functions of time isshown in FIGS. 7 and 8 for the cultivations of TN1 with ammonium andserine as nitrogen sources, respectively. In the cultivation on ammoniumthe carbon dioxide content in the exhaust gas decreased rapidly to zerowithin 40 minutes after depletion of glucose in the medium alsoformation of other products ended. Thus, the metabolism stopped when theavailable carbon source was consumed. In the cultivation with serine asnitrogen source the drop of the carbon dioxide content in the exhaustgas lasted 400 minutes after depletion of glucose. The measurementsstrongly indicated that this was due to degradation of serine topyruvate and further into acetate whereby carbon dioxide was formedsince the concentration of acetate continued to increase after depletionof glucose while the concentrations of the other products remainedconstant (FIG. 8). Due to capacity problems only the total consumptionof serine in the cultivation was measured by quantifying the serinecontent in the start and end samples. Hence, it could not be calculatedwhether the acetate formation after glucose depletion was due to serinedegradation. The slow decrease in carbon dioxide formation afterdepletion of glucose was not observed in the cultivations of TN9 andTN12 when serine was used as nitrogen source. This could indicate thatacetate was produced in the cultivation of TN1 to form NADPH, which inturn was consumed in assimilation of ammonium, stemming from degradationof serine, by the NADPH-dependent glutamate dehydrogenase.

Biomass samples were withdrawn from the batch cultivations when thecells had reached the exponential growth phase and the specificactivities of Gdh1p, Gdh2p and Glt1p were measured in the pool ofproteins extracted from these samples (Table 5).

TABLE 5 Strain TN1 TN9 TN12 TN22 Serine (mM) 0 60 0 60 0 60 0 Ammonium(mM) 60 0 60 0 60 0 60 Gdh1p 1.522 1.197 0 0 0 0 0 Gdh2p 0.020 0.2210.055 0.218 0.006 0.329 0.625 Glt1p 0.030 0.048 0.045 0.045 0.050 0.0400.040 Table 5. Specific enzyme activities of the NADPH-dependent andNADH-dependent glutamate dehydrogenases and glutamate synthase inprotein extracts from biomass samples withdrawn in the exponentialgrowth phase of strains TN1, TN9, TN12 and TN22 in the glucose-limitedbatch cultivations with either ammonium or serine as nitrogen source.

The specific activity of the NADPH-dependent glutamate dehydrogenasedecreased from 1.522 to 1.197 units per mg TCP while that of theNADH-dependent isoenzyme increased from 0.020 to 0.221 units per mg TCPwhen serine was used as nitrogen source instead of ammonium. Also asmall increase in the specific activity of glutamate synthase wasobserved. This could indicate that a small shift occurred in thecofactor specificity of ammonium assimilation into glutamate towardsconsumption of both NADPH and NADH. Since the reduction in the glycerolyield was accounted for by the effect on surplus NADH formation ofserine consumption the major part of the intracellular ammonium probablystill was assimilated under consumption of NADPH by Gdh1p.

Deletion of GDH1 in TN9 resulted in a decrease in the specific growthrate to 0.22 h⁻¹ when ammonium was used as nitrogen source. The specificactivities of Gdh2p and Glt1p in the exponential growth phase were 0.055and 0.045 units per mg TCP, respectively, while no activity of Gdh1pcould be detected. Thus, the reduction in the specific growth rate ofthe strain probably was due to a reduction in the rate of glutamatesynthesis from ammonium and 2-oxoglutarate since the total specificactivity of the two enzymes was more than ten times lower that of Gdh1pin TN1. The low activity of Gdh2p compared to the activity of the enzymein the continuous cultivations was due to transcriptional repression byglucose (Coschigano et al., 1991). As observed in the continuouscultivations assimilation of ammonium by a combination of glutamatesynthase and the NADH-dependent glutamate dehydrogenase resulted in anincrease in the ethanol yield and a decrease in the glycerol yield dueto consumption of NADH and ATP in the synthesis of glutamate as comparedto consumption of only NADPH in strain TN1. When serine was used asnitrogen source the same qualitative changes in the yields of ethanol,glycerol and acetate was observed as in the cultivations of TN1. Theincrease in the acetate yield from 0.004 c-moles per c-mole glucose to0.018 c-moles per c-mole glucose and serine was lower than observed inTN1, indicating that part of the NADPH consumed by Gdh1p in TN1 wassynthesised through oxidation of acetaldehyde to acetate by thecytoplasmic NAD(P)⁺-dependent aldehyde dehydrogenase. The decrease inthe biomass yield of TN9 was lower when serine was used as nitrogensource instead of ammonium. This was not observed in the cultivations ofTN1 and TN12 and could was not explained by any of the measured changesin product yields or enzyme activities.

The activities of Gdh2p and Glt1p in the exponential growth phase ofTN12 were 0.006 and 0.050 units per mg TCP, respectively, when ammoniumwas used as nitrogen source. Thus, the total activity of the enzymes inthe ammonium assimilation was even lower than observed in TN9 andresulted in a further reduction in the maximum specific growth rate to0.15 h⁻¹, almost a decrease to one third of μ_(max) in TN1. Sinceactivity of Gdh2p was close to zero in TN12 under these growthconditions the synthesis of glutamate was catalysed by glutamatesynthase alone. This led to a small increase in the ethanol yield. Asopposed to the observations from the cultivations of TN1 and TN9 onammonium as nitrogen source a dramatic increase in the acetate yield anda corresponding reduction in the biomass yield was observed in TN12. Thedrop in the amount of biomass synthesised per mole of glucose suggestedthat a significant change in the anabolism of the cell had occurred. Animportant role of glutamate in the metabolism is donation of ammonium insynthesis of many amino acids through deamination to 2-oxoglutaratecatalysed by the two-glutamate dehydrogenases. As described the specificactivities of these were very low in TN12, which could limit the fluxthrough the synthesis pathways to these amino acids and thus, the totalflux towards biomass synthesis. The increase in acetate formation couldbe due to an increase in NADPH consumption in biomass synthesis. Whenserine was used as nitrogen source the specific activity of Gdh2pincreased to 0.329 units per mg TCP. This represented an induction ofthe enzyme by a factor of more than 50 while the Gdh2p activity onlyincreased ten times in TN1 when serine was added. Hence, the inductionof the CHA1 promoter by serine was functional in TN12. The activity ofGlt1p decreased slightly to 0.040 units per mg TCP. The increasedactivity of the NADH-dependent glutamate dehydrogenase resulted in amarked increase in the maximum specific growth rate of the strain. Thisclearly illustrated that the growth rate of the strain was limited bythe flux towards glutamate when ammonium was used as nitrogen source.Furthermore, it demonstrated that Gdh2p was able to substitute bothGdh1p and Glt1p in synthesis of glutamate from ammonium and2-oxoglutarate. The high Gdh2p activity also resulted in a biomass yieldcomparable to the values obtained in the cultivations of TN1 and TN9.This supported the theory that the low biomass yield on ammonium was dueto a limitation in biomass synthesis by the deamination reaction fromglutamate to 2-oxoglutarate.

In order to analyse the effect of GDH2 overexpression in TN9 without theinfluence of serine addition anaerobic batch cultivations of strain TN22was carried out with glucose as carbon source and ammonium as nitrogensource. When the original GDH2 promoter was substituted by the strong,constitutive PGK promoter in TN22, a Gdh2p activity in the exponentialgrowth phase of 0.625 was obtained. This resulted in a dramatic increasein the maximum specific growth rate from 0.22 h⁻¹ to 0.39 h⁻¹ which wasvery close to μ_(max) in strain TN1. Again this illustrated that theNADH-dependent glutamate dehydrogenase could fully substitute theNADPH-dependent isoenzyme if expressed at a sufficient level. Thebiomass yield in TN22 increased by 11% compared to TN9 and was evenhigher than in TN1. This resulted in a higher glycerol yield since theamount surplus NADH formed increased per c-mole of glucose that wasconsumed. The increase in the ethanol yield that was observed in TN9 wasabsent in the cultivation of TN22, which illustrated that glutamatesynthesis from ammonium and 2-oxoglutarate, occurred without consumptionof ATP in TN22.

Concluding remarks. The work described herein above was carried out toanalyse if the activity of the GDH1 encoded NADPH-dependent glutamatedehydrogenase could advantageously be substituted by the GDH2 encodedNADH-dependent isoenzyme in a process involving assimilation of ammoniaand 2-oxoglutarate into glutamate. Another objective was to analyse theeffect of such metabolically engineered ammonia assimilation on theproduct formation in anaerobic cultivations.

The anaerobic batch cultivation of strain TN22 clearly demonstrated thathigh expression of GDH2 in a Δgdh1 mutant resulted in a strain whereinthe physiological role of Gdh1p in a wild-type strain had been takenover by Gdh2p. Furthermore, it was shown that this change in themetabolism resulted in a reduction of glycerol formation. Thus,formation of surplus NADH in biomass synthesis exerts a major effect onglycerol synthesis. Most likely, a further reduction in the formation ofundesirable metabolic products can be obtained by metabolicallyengineering other reactions catalysed by isoenzymes with differentcofactor specificities. The reduced glycerol yield did not result in anincrease of the metabolic flux towards ethanol. Accordingly, in themetabolically engineered strains, the formation of ethanol is notlimited by the flux of metabolites towards other fermentation products.

An attempt was made to study the effect of GDH2 overexpression on theanaerobic physiology in more detail by introducing the induciblepromoter of CHA1 in front of GDH2 on chromosome IV. The induction of thepromoter by serine was clearly evident, but very high amounts of theamino acid had to be added to the medium in order to obtain a highexpression of GDH2. Addition of up to 30 mM of serine to the medium in acontinuous cultivation affected the metabolism of the cells andcomplicated the analysis of the experiments. In batch cultivations ofTN12, induction of GDH2 expression by serine resulted in a significantincrease in the specific growth rate of that strain. This resultreiterated the conclusion reached herein above that the activity ofGdh2p can indeed substitute that of Gdh1p in a cellular metabolism.

A significant increase in the formation of ethanol and a decrease in theformation of glycerol were observed, when GDH1 was deleted in strainTN9. This may be due to a synthesis of glutamate via two coupledreactions mediated by GLT1 encoded glutamate synthase and GLN1 encodedglutamine synthetase. Synthesis of glutamate via these two coupledreactions results in consumption of both NADH and ATP and saidconsumption may explain the observed changes in product formation.Unfortunately, the maximum specific growth rate of strain TN9 was alsoreduced by the deletion in question and thus, no overall increase inethanol productivity was obtained. The lower growth rate was probablydue to a limitation in the synthesis rate of glutamate. This isconceivable since the activity of Glt1p was much lower than the activityof Gdh1p which catalysed glutamate formation in strain TN1.

The experiments described above have demonstrated that the observedgrowth defect could be alleviated by an overexpression of GDH2.Accordingly, a key question is: Does an overexpression of Glt1p andGln1p result in a strain with an increased ethanol productivity as wellas a higher growth rate, as compared to a wild-type strain?

EXAMPLE 2 Metabolically Engineered Ammonia Assimilation and EthanolProduction in Yeast

Introduction

In this example, a new strategy is presented for optimisation of theethanol yield in Saccharomyces cerevisiae through metabolic engineering.It is based on the physiological roles of glycerol and ethanol inoxidation of surplus NADH and in formation of ATP, respectively, underanaerobic growth conditions. Experimental results from anaerobic batchcultivations of strains developed on the basis of the strategy arepresented.

Materials and Methods

Microorganisms and their maintenance. All Saccharomyces cerevisiaestrains were generated from Saccharomyces cerevisiae T23D. The strainwas kindly provided by Jack Pronk from the Department of Microbiologyand Enzymology, Kluyver Laboratory of Biotechnology, Delft University ofTechnology, The Netherlands. The yeast strains were maintained at 4° C.on YPG agar plates, monthly prepared from a lyophilised stock kept at−80° C. Escherichia coli DH5α (F—F80dlacZ DM 15 D(lacZYA-argF) U 169deoR recA1 endA1 hsdR17(r_(k) ⁻m_(k) ⁺) supE44l⁻ thi-1 gyra96 relA1)(GIBCO BRL. Gaithersburg, Md., USA) was used for subcloning.

Preparation of DNA. Plasmid DNA from E. coli was prepared with Qiagencolumns (Qiagen GmbH, Düsseldorf, Germany) following the manufacturer'sinstructions. For the purification of DNA fragments used for cloningexperiments, the desired fragments were separated on 0.8% agarose gels,excised and recovered from agarose using the Qiagen DNA isolation kit(Qiagen GmbH, Düsseldorf, Germany). Chromosomal DNA from Saccharomycescerevisiae was extracted as follows. Cells were cultivated in medium inshake flasks and harvested at OD=1.5, 10 mg of wet cells wereresuspended in 0.5 ml Tris-Cl (pH 8.0) and quenched with 0.5 ml glassbeads (size 250–500 microns) in the presence of 0.5 ml Tris-saturatedphenol (pH 8.0). The DNA was extracted from the phenol phase withchloroform, precipitated with 98% ethanol and resuspended in TE buffer.RNA in the extract was removed by treatment with RNAaseA (purchased fromPromega) and finally the DNA was purified by precipitation withethanol/lithium chloride and resuspended in TE buffer. The DNA primerswere purchased from DNA Technology (Aarhus, Denmark).

Overexpression of GLT1. Primers Glt1start (5′-GCG CGG GAT CCT CTA GAATGC CAG TGT TGA AAT CAG AC-3′), SEQ ID NO:9, containing restrictionenzyme sites for BamHI and XbaI in front of nucleotides 1 to 21 of GLT1,and Glt1stop (5′-CGC GCG GAT CCC CGC GGG CTG GAC CAT CCC AAG GTT CC-3′).SEQ ID NO:10, containing restriction enzyme sites for BamHI and SacII infront of nucleotides 1149 to 1169 of GLT1, were used to clone parts ofthe structural gene of GLT1 by PCR with the pfu polymerase (New EnglandBiolabs). A DNA fragment of the correct size was isolated from a 0.8%agarose gel after electrophoresis and digested with BamHI overnight. Thefragment was ligated into the unique BglII digestion site of plasmidYep24-pPGK behind the PGK promoter and in front of the PGK terminator(Walfridsson et al. 1997), resulting in plasmid Yep24-pPGK-GLT1. A 2.5kb SmaI/SacII DNA fragment, consisting of the PGK promoter and thecloned part of GLT1 was isolated from Yep24-pPGK-GLT1. The fragment wasligated into plasmid pFA6A-kanMX3 (Wach et al. 1994), digested withEcoRV and SacII, resulting in plasmid pPGK-GLT1 (FIG. 9). The plasmidwas linearised by digestion with EcoRV prior to transformation. Correctinsertion of the plasmid into the GLT1 locus on chromosome IV wasverified by PCR analysis of chromosomal DNA extracted from transformantswith resistance towards geniticin. For this purpose primers PGKverif,spanning the region 420 to 400 bp upstream of the PGK start codon, andGLT1verif, spanning the region from nucleotides 124, to 1260 of GLT1,were constructed. Loop out of the geniticin resistance gene byhomologues recombination of the two direct repeats flanking the gene wasobtained by cultivating correct transformants for up to 100 generationsin non-selective YPD medium followed by plating of approximately 50000colonies on YPD-plates. The colonies were then replica plated to YPDplates containing 150 mg per liter geniticin and transformants withoutresistance towards geniticin were isolated. The loop out frequency wasapproximately 1 per 25000 colonies. It was verified by PCR analysis ofchromosomal DNA from loop out transformants that the PGK promoter wasstill introduced in front of GLT1.

Overexpression of GLN1. Primers Gln1start (5′-GCG CGG GAT CCT CTA GAATGG CTG AAG CAA GCA TCG AA-3′), SEQ ID NO: 11, containing restrictionenzyme sites for BamHI and XbaI in front of nucleotides 1 to 21 of GLN1,and Gln1stop (5′-CGC GCG GAT CCC CGC GGT TAT GAA GAT TCT CTT TCA AA-3′).SEQ ID NO:12, containing restriction enzyme sites for BamHI and SacII infront of nucleotides 1093 to 1113 of GLN1, were used to clone GLN1 byPCR with the pfu polymerase (New England Biolabs). The obtained DNAfragment was used to construct plasmid pPGK-GLN1, containing GLN1 behindthe promoter of PGK inserted into pFA6A-kanMX3 (FIG. 9), as describedabove for plasmid pPGK-GLT1, pPGK-GLN1 was linearised by digestion withKpnI prior to transformation. Correct insertion of the plasmid into theGLN1 locus on chromosome XVI was verified by PCR analysis of chromosomalDNA extracted from transformants with resistance towards geniticin. Forthis purpose primers PGKverif, spanning the region 420 to 400 bpupstream of the PGK start codon, and GLN1verif, spanning the region fromnucleotides 52 to 70 downstream of GLT1, were constructed.

Transformation of E. coli and S. cerevisiae. E. coli DH5α wastransformed by electro-transformation using the Bio-Rad electroporationequipment (Biorad Laboratories, Richmond, USA). Transformants wereselected on L broth plates containing 100 mg/ml ampicillin. S.cerevisiae cells were made competent for plasmid uptake by treatmentwith lithium acetate and polyethyleneglycol (Schiestl & Gietz, 1989). 3μg of DNA was used for each transformation. Transformants were suspendedin YPD for 24 hours prior to plating on YPD, containing 150 mg geniticinper liter, in order to obtain expression of the G418 resistance gene.

Medium in the batch cultivations. The strains of S. cerevisiae werecultivated in a mineral medium prepared according to Verduyn et al.(1990). Vitamins were added by sterile filtration following heatsterilisation of the medium. The concentrations of glucose and (NH₄)₂SO₄initially in the batch cultivations were 25 g per l and 3.75 g per l,respectively. Growth of S. cerevisiae under anaerobic conditionsrequires the supplementary addition to the medium of ergosterol andunsaturated fatty acids, typically in the form of Tween 80 (Andreasen &Stier, 1953: Libudzisz et al., 1986). Ergosterol and Tween 80 weredissolved in 96% (v/v) ethanol and the solution was autoclaved at 121°C. for 5 min. The final concentrations of ergosterol and Tween 80 in themedium were 4.2 mg per g DW and 175 mg per g DW, respectively. Toprevent foaming 75 μl per l antifoam (Sigma A-5551) was added to themedium.

Experimental set-up for the batch cultivations. Anaerobic batchcultivations were performed at 30° C. and at a stirring speed of 600 rpmin in-house manufactured bioreactors. The working volume of the batchreactors was 4.5 liters. pH was kept constant at 5.00 by addition of 2 MKOH. The bioreactors were continuously sparged with N₂ containing lessthan 5 ppm O₂, obtained by passing N₂ of a technical quality (AGA 3.8),containing less than 100 ppm O₂, through a column (250×30 mm) filledwith copper flakes and heated to 400° C. The column was regenerateddaily by sparging it with H₂ (AGA 3.6). A mass flow controller(Bronkhorst HiTec F201C) was used to keep the gas flow into thebioreactors constant at 0.50 l nitrogen min⁻ liter⁻ Norprene tubing(Cole-Parmer Instruments) was used throughout in order to minimisediffusion of oxygen into the bioreactors. The bioreactors wereinoculated to an initial biomass concentration of 1 mg l⁻¹ withprecultures grown in unbaffled shake flasks at 30° C. and 100 rpm for 24hours. Ethanol evaporation from the bioreactors was minimised by off-gascondensers cooled to 2° C. The anaerobic batch cultivations of strainsTN1, TN9, TN15, TN17 and TN19 were each carried out three times withidentical results.

Determination of dry weight. Dry weight was determined gravimetricallyusing nitrocellulose filters (pore size 0.45 μm: Gelman Sciences). Thefilters were predried in a microwave oven (Moulinex FM B 935Q) for 10min. A known volume of culture liquid was filtered and the filter waswashed with an equal volume of demineralised water followed by drying ina microwave oven for 15 min. The relative standard deviation (RSD) ofthe determinations was less than 1.5% based on triple determinations(n=3).

Analysis of medium compounds. Cell-free samples were withdrawn directlyfrom the bioreactor through a capillary connected to a 0.45 μm filter.Samples were subsequently stored at −40° C. Glucose, ethanol, glycerol,acetic acid, pyruvic acid, succinic acid and 2-oxoglutarate weredetermined by HPLC using an HPX-87H Aminex ion exclusion column(RSD<0.6%, n=3). The column was eluted at 60° C. with 5 mM H₂SO₄ at aflow rate of 0.6 ml min⁻¹. Pyruvic acid, acetic acid and 2-oxoglutaratewere determined with a Waters 486 UV meter at 210 mm whereas the othercompounds were determined with a Waters 410 refractive index detector.The two detectors were connected in series with the UV detector first.The CO₂ concentration in the off-gas was determined using a Brüel & Kjær1308 acoustic gas analyser (RSD=0.02%) (Christensen et al., 1995). In aseparate experiment the off gas from the bioreactor was bubbled throughliquid nitrogen and the ethanol concentration in the frozen mixture ofwater, ethanol and acetaldehyde was determined by HPLC after evaporationof the N₂. Hereby the loss of ethanol through the reflux condenser ofthe bioreactor was determined to be between 4% and 9% of the ethanolformed by the bioreaction depending on the dilution rate (Schulze,1995). In the carbon balances the measured ethanol fluxes were correctedfor this loss through evaporation.

Measurement of enzyme activities. Culture liquid was withdrawn from thebioreactor into an ice cooled beaker, centrifuged and washed twice with10 mM potassium phosphate buffer (pH 7.5, 2° C.) containing 2 mM EDTA.Subsequently the cells were resuspended in 4.2 ml 100 mM potassiumphosphate buffer (pH 7.5, 2° C.) containing 2 mM MgCl₂ followed byimmediate freezing in liquid nitrogen and storage at −40° C. Prior toanalysis 0.22 ml of 20 mM DTT was added to the samples whereafter theywere distributed into precooled 2 ml eppendorf tubes containing 0.75 mlglass beads (size 0.25–0.50). The cells were disrupted in a bead millfor 12.5 min. (0° C.). The test tubes were centrifuged (20000 rpm. 20min., 0° C.) whereafter the supernatants were pooled in one test tube.During the following analyses the extract was kept on ice. Enzyme assayswere performed at 30° C. using a Shimadzu UV-260 spectrophotometer at30° C. Reaction rates, corrected for endogenous rates, were proportionalto the amount of extract added. All enzyme activities are expressed asmicromole of substrate converted per minute per mg total cellularprotein as determined by the Lowry method. Glutamate dehydrogenase (NAD⁺and NADP⁺) (EC 1.4.1.5 and EC 1.4.1.4, respectively) were assayed asdescribed by Bruinenberg et al. (1983a). Glutamine synthetase (EC6.3.1.2) and glutamate synthase (GOGAT) (EC 1.4.1.14) was assayed asdescribed by Holmes et al. (1989).

Results

Metabolic control analysis. Anaerobic physiology in continuouscultivations of S. cerevisiae CBS8066 has previously been analysed hymeans of metabolic flux analysis and said theoretical althoughpractically applicable analysis showed that the flux through thereaction catalysed by the NADPH-dependent glutamate dehydrogenase(reaction 25 in a proposed model) was 8.0 c-mmoles per g biomass perhour at a dilution rate of 0.3 h⁻¹, although the net flux from2-oxoglutarate to glutamate was only 2.0 c-mmoles per g biomass perhour.

Accordingly, if Glt1p and Gln1p catalysed the same reaction instead ofGdh1p, one mole of NADH and one mole of ATP would be expected to beconsumed per mole of glutamate synthesised, instead of consumption ofone mole NADPH. The result of such a metabolic engineering wouldconsequently be a reduction in a surplus formation of NADH.

A decrease in the formation of glycerol from 9.8 to 5.0 c-mmoles per gbiomass per hour, a reduction by 49%, was predicted. Furthermore, theconsumption of ATP was hypothesised to result in a 6% increase ofethanol formation, from 54.9 to 58.2 c-mmoles per g biomass per hour.

Construction of new strains. Earlier the haploid S. cerevisiae strainTN1 was constructed from a diploid progeny of the industrial modelstrain S. cerevisiae CBS8066 (Nissen et al., 1998b). This was done bydeletion of HO, encoding an endonuclease involved in mating typeswitching, and isolation of stable haploids following sporulation ofdeletion mutants. Furthermore GDH1 was deleted in TN1 as describedearlier, resulting in strain TN9 (Nissen et al., 1998a). In this studythe object was to construct new strains from TN9 with a stable,constitutive overexpression of GLN1, encoding glutamine synthetase, andGLT1, encoding glutamate synthase. This was achieved by inserting thestrong, constitutive promoter of PGK in front of the two genes onchromosomes XVI and IV, respectively. PGK encodes one of the mostabundant mRNA and protein species in the cell, accounting for 1% to 5%of the total cellular mRNA and protein during growth on fermentativecarbon sources (Dobson et al. 1982). By integrating the promoter intothe chromosome, problems with plasmid loss, resulting in an unstablephenotype, could be avoided. TN9 was transformed with either of thelinearised plasmids pPGK-GLN1 and pPGK-GLT1, resulting in strains TN15and TN17, respectively. It was verified by PCR analysis that the strong,constitutive promoter of PGK had been inserted into the chromosome infront of the structural genes, encoding glutamine synthetase andglutamate synthase, respectively. TN17 was cultivated in non-selectiveYPD medium for 100 generations in order to remove the resistance againstgeniticin by homologues recombination of the direct repeats flanking theresistance gene in the chromosome. This resulted in isolation of strainTN18 which had lost the geniticin resistance but maintained the strongpromoter in front of GLT1. TN18 was transformed with pPGK-GLN1 asdescribed above, resulting in strain TN19 with the PGK promoter insertedin front of both GLN1 and GLT1 in the chromosome. Table 6 lists thephenotypes of all strains that were cultivated in batch reactors in thisstudy.

TABLE 6 Strain Phenotype Reference TN1 MATα ho-Δ1 Nissen et al., 1998bTN9 MATα ho-Δ1 ura3-Δ20::SUC2 gdh1-Δ1:: Nissen et al., 1998a URA3 TN15MATα ho-Δ1 ura3-Δ20::SUC2 gdh1-Δ1:: This study URA3 gln1::(PGKp-GLN1)TN17 MATα ho-Δ1 ura3-Δ20::SUC2 gdh1-Δ1:: This study URA3glt1::(PGKp-GLT1) TN19 MATα ho-Δ1 ura3-Δ20::SUC2 gdh1-Δ1:: This studyURA3 glt1::(PGKp-GLT1) gln1::(PGKp- GLN1) Table 6. Phenotypes of thestrains cultivated in anaerobic batch fermentations in this study.

Glucose limited batch cultivations. The anaerobic physiology of thegenetically engineered S. cerevisiae strains were studied in batchcultivations with glucose as the primary carbon source and ammoniumnitrogen source. This was done to quantify the effect of the geneticchanges on the specific enzyme activities of Gdh1p, Gdh2p, Glt1p andGln1p, the maximum specific growth rate, μ_(max), and on the productyields.

The specific enzyme activities were measured in protein extracts frombiomass samples withdrawn from the bioreactors when the cells were inthe exponential growth phase (Table 7).

TABLE 7 TN1 TN9 TN15 TN17 TN19 Gdh1p 1.522 n.d. n.d. n.d. n.d. Gdh2p0.020 0.055 0.045 0.028 0.033 Gln1p 0.011 0.009 0.068 0.009 0.062 Glt1p0.030 0.045 0.045 0.195 0.211 Table 7. Product yields of strains TN1,TN9, TN13, TN17 and TN19 in the anaerobic, glucose-limited batchcultivations that were carried out in this study. Unit: c-moles productper c-mole glucose.

As observed earlier the activity of the NADPH-dependent glutamatedehydrogenases in strain TN1 were 50–100 times higher than the remainingenzymes involved in assimilation of ammonium. This demonstrated theimportance of this enzyme in wild-type cells during growth on ammoniumas nitrogen source. No activity of Gdh1p could be detected in extractsfrom the four strains where GDH1 had been deleted. Thus, GDH3p had nophysiological role in this strain background when ammonium was used asnitrogen source. The activity of the NADH-dependent glutamatedehydrogenase, encoded by GDH2, was approximately 2.5 times higher instrain TN9 than observed in the haploid wild-type. As described earlierthis increase probably was due to a decrease in the intracellularconcentration of glutamine since this metabolite represses expression ofGDH2 at the transcriptional level (Nissen et al., 1998a). This decreasecould be due to a limitation in the synthesis rate of glutamate in cellswith a deletion in GDH1. Almost a similar increase in Gdh2p activity wasobserved in strain TN15, indicating that overexpression of thestructural gene for glutamine synthetase only resulted in a limitedincrease in the intracellular concentration of glutamine. In strainsTN17 and TN19 the Gdh2p activity was reduced to a level close to thatobserved in TN1. Thus, an increase in the intracellular glutamineconcentration to wild-type probably was achieved by overexpression ofglutamate synthase, resulting in an increase in the synthesis rate ofglutamate. Insertion of the strong constitutive promoter of PGK in frontof GLN1 into the chromosome of TN15 and TN19 resulted in an increase inthe specific activity of glutamine synthetase from approximately 0.0100units per mg total cellular protein (TCP) in strains TN1 and TN9 to0.068 and 0.062 units per mg TCP, respectively. Insertion of thepromoter in front of GLT1 resulted in a five-fold increase in theactivity of glutamate synthase in strains TN17 and TN19 compared to thethree other strains. Thus, it was concluded that the new promoter hadbeen inserted correct into the chromosome and that this resulted in theexpected increase in the specific activities of glutamine synthetase andglutamate synthase.

In FIG. 10 the production of biomass in the exponential growth phases ofTN1, TN9, TN17 and TN19 as functions of time are depicted. Deletion ofGDH1 resulted in a reduction in the maximum specific growth rate,μ_(max), from 0.41 h⁻¹ in strain TN1 to 0.21 h⁻¹ in strain TN9 (FIG.10). As mentioned earlier this probably was due to a reduction in thesynthesis rate of glutamate since the total specific activities of theenzymes that potentially could substitute the physiological role ofGdh1p, Gdh2p and Glt1p, was 15 times lower than the specific activity ofGdh1p in TN1. Overexpression of GLN1 in TN9, resulting in strain TN15,gave only a small increase in the maximum specific growth rate, to 0.24h⁻¹ (results not shown). This very limited effect of the increase inGln1p activity was probably due to a slightly higher flux towardssynthesis of glutamine. As described earlier glutamine is one of thesubstrates in the reaction, catalysed by glutamate synthase, whereinglutamate is formed when GDH1 is deleted. Overexpression of GLN1 in TN15probably removes a limitation in the glutamine supply to the reactioncatalysed by Gltp, which results in the observed increase in μ_(max)Overexpression of GLT1 had a significant effect on the maximum specificgrowth rate of TN17. An increase to 0.31 h⁻¹ was observed. This clearlydemonstrated that the reduction in the growth rate of TN9 was caused bya low synthesis rate of glutamate and that this limitation could bepartly removed by constructing a strain with a high specific activity ofglutamate synthase. Overexpression of both GLT1 and GLN1 was obtained inTN19. This led to a further increase in the maximum specific growth rateto 0.37 h⁻¹. Thus, the increase in the specific activity of glutaminesynthetase had a more pronounced effect on the specific growth rate in astrain where the specific activity of glutamate synthase was highcompared to in a strain with a wild-type level of activity. Thisindicated that overexpression of GLT1 alone probably resulted indepletion in the intracellular pool of glutamine, which limited theeffect of the increase in the specific enzyme activity of Glt1p in TN17.This limitation was apparently removed by overexpression of thestructural gene encoding glutamine synthetase.

The consumption of glucose and the production of ethanol, glycerol,acetate, pyruvate, succinate, biomass and carbon dioxide were measuredin filtered samples withdrawn from the batch cultivations of TN1, TN9,TN15, TN17 and TN19. No variations between the five strains in formationof the organic acids were detected and thus, only the total yield ofthese metabolites is listed in Table 8.

TABLE 8 TN1 TN9 TN15 TN17 TN19 Ethanol 0.480 0.520 0.521 0.530 0.531Glycerol 0.097 0.060 0.059 0.061 0.060 Biomass 0.121 0.114 0.114 0.1100.110 Carbon dioxide 0.261 0.275 0.272 0.271 0.273 Organic acids 0.0090.009 0.010 0.009 0.008 Total 0.968 0.978 0.976 0.981 0.982 Table 8. Thespecific activities of the NADPH-dependent and NADH-dependent glutamatedehydrogenases, glutamine synthetase and glutamate synthase in proteinextracts from biomass samples withdrawn in the exponential growth phasesof strains TN1, TN9, TN15, TN17 and TN19 in the anaerobic,glucose-limited batch cultivations.

FIGS. 11, 12 and 13 show the consumption of glucose and the productionof ethanol, glycerol and carbon dioxide as functions of time in thecultivations of strains TN1, TN9 and TN 19, respectively. In allcultivations formation of ethanol, glycerol and carbon dioxide stoppedimmediately after depletion of glucose in the medium. No consumption ofthe products was detected as long as the bioreactors were sparged withnitrogen, which demonstrated that anaerobic growth conditions had beenobtained in the cultivations. The reduction in μ_(max) of TN9 resultedin an increase in the duration of the anaerobic fermentation of thecarbon source of 3.5 hours compared to TN1. This elongation of thefermentation time was significantly reduced in strain TN19. Here thefermentation lasted 40 minutes longer than observed in the cultivationsof TN I. A relative increase in the ethanol yield of 8% was measured inthe cultivations of TN9 compared to TN1 while the relative decrease inthe glycerol yield was measured to 38% (Table 8). As described earlierthis was due to formation of glutamate by the NADH and ATP consumingreactions catalysed by glutamate synthase and glutamine synthetase inTN9 compared to formation of glutamate by the NADPH consuming reactioncatalysed by glutamate dehydrogenase I in TN1. The increase in ATPconsumption in biomass synthesis resulted in a reduction of the biomassyield in TN9, by 6%. Overexpression of GLN1 alone had no significantinfluence on the product formation of strain TN15 compared to TN9. Thefive-fold increase in the specific glutamate synthase activity that wasobtained by overexpression of GLT1 in strain TN17 resulted in a smallincrease in the ethanol yield and a similar small increase in biomassformation. This indicated that the total flux through the reactionscatalysed by Glt1p and Gln1p probably was slightly higher in TN17compared to TN9 which resulted in an increase in the ATP cost of biomasssynthesis. No decrease in glycerol formation was detected. Thus, thedifference in ethanol formation of TN17 and TN9 could be an artefactcaused by small errors in the measurement of ethanol. The standarddeviation in the ethanol yields obtained in anaerobic cultivations ofthe same strain was 3.5–4.1%. Overexpression of both GLN1 and GLT1 instrain TN19 did not results in any changes in product formation comparedto TN17.

EXAMPLE 3 Expression of a Transhydrogenase Activity in Lactococcuslactis and Escherichia coli

Introduction

cth, encoding the cytoplasmic transhydrogenase from Azotobactervinelandii, was cloned by PCR using primers BglII-cth(5′-tacgaagatctGCTGTATATAAcTACGATGTGGTGG-3′) (SEQ ID NO:13) and CTH-XhoI(5′-tagcactcgagttaAAAAAGCCGATTGAGACC-3′) (SEQ ID NO:14) and pfupolymerase. The resulting DNA fragment was digested with the restrictionenzymes BglII and XhoI and inserted into the multi cloning site of theE. coli/L. lactis shuttle vector pTRKH2-p170 behind a strongconstitutive derivate of the promoter p170 (S. M. Madsen, J. Arnau, A.Vrang, M. Givskov and H. Israelsen (1999). Molecular Microbiology 32,75–87). The resulting plasmid was denoted pTRKH2-p 170-cth. The promoterregion of the vector and the inserted cth were sequenced, whereby it wasverified that the gene had been inserted correctly into the shuttlevector.

E. coli DH5α and L. lactis subsp. cremoris were both transformed withpTRKH2-p170-cth and transformants were selected on plates containingcomplex medium (LB and GM13, respectively) supplemented witherythromycin. Independent pTRKH2-p170-cth transformants of both E. coliand L. lactis were grown in shake flasks in LB medium and GM13 medium,respectively, supplemented with erythromycin. In the late exponentialgrowth phase, cell samples were withdrawn from the shake flasks and theprotein pools of the cells were extracted.

The extracted protein pools were assayed for activity of the cytoplasmictranshydrogenase. The results are listed below

Transhydrogenase activity Microorganism (U per mg protein) E. coli DH5αnot detectable E. coli DH5α pTRKH2-p170-cth 0.568 L. lactis subsp.cremoris not detectable L. lactis subsp. cremoris pTRKH2-p170-cth 0.107

From the data it was concluded that the cytoplasmic transhydrogenase hadbeen succesfully expressed in both E. coli and L. lactis.

Saccharomyces cerevisiae strains were deposited under the BudapestTreaty with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1b, D-38124 Braunschweig GERMANY, as follows

Strain Deposit No. Deposit Date TN4 DSM 12267 Jun. 19, 1998 TN15 DSM12274 Jun. 24, 1998 TN17 DSM 12275 Jun. 24, 1998 TN19 DSM 12276 Jun. 24,1998 TN22 DSM 12277 Jun. 24, 1998

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1. A recombinant yeast cell comprising: i) at least one increasedexpressible yeast enzyme activity controlling anabolic metabolism ofammonia in said cell as a nutrient source, said increased enzymeactivity being selected from the group consisting of: (a) a glutamatedehydrogenase activity catalyzing the reaction: 2-oxoglutarate+NH₄⁺+NADH→glutamate+NAD⁺ and being that encoded by GDH2 of Saccharomycescerevisiae; (b) a glutamate synthase activity catalyzing the reaction:2-oxoglutarate+glutamine+NADH→2 glutamate+NAD⁺ and being that encoded byGLT1 of Saccharomyces cerevisiae; and (c) a glutamine synthetaseactivity catalyzing the reaction: glutamate+NH₄ ⁺+ATP→glutamine+ADP+Piand being that encoded by GLN1 of Saccharomyces cerevisiae; wherein eachincreased enzyme activity is that of (1) an enzyme, endogenous to saidcell, encoded by a nucleic acid coding sequence operably linked to atleast one regulatory sequence not natively associated with said nucleicacid coding sequence, whose expression is increased as compared to theexpression of the enzyme activity when said nucleic acid coding sequenceis associated with its native regulatory sequence or (2) an enzyme,exogenous to said cell, encoded by a nucleic acid coding sequence,operably linked to at least one regulatory sequence, and wherein therecombinant yeast cell is further characterized by ii) a reducedexpressible enzyme activity controlling anabolic metabolism of ammoniain said cell as a nutrient source, said reduced enzyme activity being anatively present NADPH-dependent glutamate dehydrogenase activity whichreduced activity is reduced compared to the native level of activity. 2.The yeast cell of claim 1, said cell comprising an increased enzymeactivity (b) and an increased enzyme activity (c).
 3. The yeast cell ofclaim 1, said cell comprising a further increased expressible enzymeactivity, said further expressible enzyme activity being the pyridinenucleotide transhydrogenase activity encoded by CTH of Azobactervinelandii as harboured by Saccharomyces cerevisiae TN4 deposited underDSM Accession No. 12267 and being operably linked to a regulatorysequence not natively associated with said further enzyme activity insaid yeast cell.
 4. The yeast cell of claim 3 wherein said cellcomprises no endogenous transhydrogenase activity.
 5. The yeast cell ofclaim 1, comprising an increased enzyme activity (b).
 6. The yeast cellof claim 5, wherein said GLT1 is that of the strain of Saccharomysescerevisiae deposited under DSM Accession No.
 12275. 7. The yeast cell ofclaim 1, comprising an increased enzyme activity (c).
 8. The yeast cellof claim 7, wherein said GLN1 is that of the strain of Saccharomycescerevisiae deposited under DSM Accession No.
 12274. 9. The yeast cell ofclaim 1, comprising an increased enzyme activity (a).
 10. The yeast cellof claim 9, wherein said GDH2 is that of the strain of Saccharomycescerevisiae deposited under DSM Accession No.
 12277. 11. The yeast cellof claim 1 in which said reduced enzyme activity is the result ofdeletion of at least part of the native nucleic acid coding sequence forthe NADPH-dependent glutamate dehydrogenase, and/or of at least part ofat least one native regulatory sequence associated with the nativenucleic acid coding sequence.
 12. The yeast cell of claim 1, which is agenetically modified Saccharomyces, Schizosaccharomyces or Pichia yeast.13. The yeast cell of claim 1, said cell being Saccharomyces cerevisiaeTM 19 as deposited under Accession No. DSM
 12276. 14. The yeast cell ofclaim 1, said cell being Saccharomyces cerevisiae TN22 as depositedunder Accession No. DSM
 12277. 15. The yeast cell of claim 1, in theform of a frozen or freeze-dried preparation.
 16. The yeast cell ofclaim 15, said cell being partly or wholly reconstitutable.
 17. Acomposition comprising the yeast cell according to claim 1, in acarrier.
 18. The composition of claim 17, wherein the carrier is aphysiologically acceptable carrier.
 19. The composition of claim 17,said composition being a fermentation starter culture.
 20. Therecombinant yeast cell of claim 1, wherein production of a firstmetabolite is substantially increased as compared to a yeast cell whichis identical to the recombinant yeast cell except that it lacks themodifications set forth in (i) and (ii).
 21. The recombinant yeast cellof claim 20, wherein said production of said first metabolite isincreased by a factor of at least 1.08.
 22. The recombinant yeast cellof claim 20, wherein said first metabolite is ethanol.
 23. Therecombinant yeast cell of claim 20, further producing a secondmetabolite, the production of said metabolite being substantiallydecreased as compared to the production of said second metabolite in ayeast cell which is identical to the recombinant yeast cell except thatit lacks the modifications set forth in (i) and (ii).
 24. Therecombinant yeast cell of claim 23, wherein said second metabolite isglycerol.
 25. A method of producing a first metabolite, said methodcomprising the steps of: i) cultivating a yeast cell according to claim20 in a suitable growth medium and under such conditions that said yeastcell produces said first metabolite; and, optionally, ii) isolating saidfirst metabolite in a suitable form; and, further optionally, iii)purifying said isolated first metabolite.
 26. A method of constructing arecombinant yeast cell according to claim 20, said method comprising thesteps of: 1) operably linking a first nucleotide sequence encoding anenzyme mediating said increased expressible enzyme activity with anexpression signal not natively associated with said first nucleotidesequence; and 2) operably linking a second nucleotide sequence encodingan enzyme mediating for said reduced expressible enzyme activity with aregulatory sequence not natively associated with said second nucleotidesequence, said regulatory sequence generating a reduced expression ofsaid second nucleotide sequence.
 27. The yeast cell of claim 1 in whichsaid at least one regulatory sequence not natively associated with saidcoding sequence is a promoter.
 28. The yeast cell of claim 1 in whichthe reduced enzyme activity is an eliminated activity.
 29. The yeastcell of claim 1 in which at least one said increased enzyme activity isthat of an enzyme endogenous to the cell.
 30. The yeast cell of claim 29in which said endogenous enzyme is encoded by its native codingsequence.
 31. The yeast cell of claim 1 in which said reduced enzymeactivity is the result of operably linking at least one regulatorysequence, not natively associated with the coding sequence encoding thenative NADPH-dependent glutamate dehydrogenase of (ii), with said codingsequence, so that expression of said native enzyme is reduced.
 32. Theyeast cell of claim 1 in which said reduced enzyme activity is theresult of repression of expression.
 33. The recombinant yeast cell ofclaim 1, comprising: at least one increased expressible yeast enzymeactivity controlling anabolic metabolism of ammonia in said cell as anutrient source, said increased enzyme activity being selected from thegroup consisting of: (a) a glutamate dehydrogenase activity catalyzingthe reaction: 2-oxoglutarate+NH₄ ⁺+NADH→glutamate+NAD⁺ and being thatencoded by GDH2 of the strain of Saccharomyces cerevisiae depositedunder DSM Accession No. 12277; (b) a glutamate synthase activitycatalyzing the reaction: 2-oxoglutarate+glutamine+NADH→2 glutamate+NAD⁺and being that encoded by GLT1 of the strain of Saccharomyces cerevisiaedeposited under DSM Accession No. 12275; and (c) a glutamine synthetaseactivity catalyzing the reaction: glutamate+NH₄ ⁺+ATP→glutamine+ADP+Piand being that encoded by GLN1 of the strain of Saccharomyces cerevisiaedeposited under DSM Accession No. 12274; wherein each increased enzymeactivity is that of (1) an enzyme, endogenous to said cell, encoded by anucleic acid coding sequence operably linked to at least one regulatorysequence not natively associated with said nucleic acid coding sequence,whose expression is increased as compared to the expression of theenzyme activity when said nucleic acid coding sequence is associatedwith its native regulatory sequence, or (2) an enzyme, exogenous to saidcell, encoded by a nucleic acid coding sequence, operably linked to atleast one regulatory sequence, and wherein the recombinant yeast cell isfurther characterized by (ii) a reduced expressible enzyme activitycontrolling anabolic metabolism of ammonia in said cell as a nutrientsource, said reduced enzyme activity being a natively presentNADPH-dependent glutamate dehydrogenase activity which reduced activityis reduced compared to the native level of activity.